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This page intentionally left blank
Preface
With this 15th edition, funqueira's Basic Histology continues as
the preeminent source of concise yet thorough information
on human tissue structure and function. For over 45 years
this educational resource has met the needs of learners for a
well-organized and concise presentation of cell biology and
histology that integrates the material with that ofbiochemistry,
immunology, endocrinology, and physiology and provides
an excellent foundation for subsequent studies in pathology.
The text is prepared specifically for students of medicine
and other health-related professions, as well as for advanced
undergraduate courses in tissue biology. As a result of its value
and appeal to students and instructors alike, Junqueira's Basic
Histology has been translated into a dozen different languages
and is used by medical students throughout the world.
Unlike other histology texts and atlases, the present
work again includes with each chapter a set of multiple-
choice Self-Test Questions that allow readers to assess their
comprehension and knowledge of important points in that
chapter. At least a few questions in each set utilize clinical
vignettes or cases to provide context for framing the medical
relevance ofconcepts in basic science, as recommended by the US
National Board of Medical Examiners. As with the last edition,
each chapter also includes a Summary of Key Points designed to
guide the students concerning what is clearly important and what
is less so. Summary Thbles in each chapter organize and condense
important information, further facilitating efficient learning.
Each chapter has been revised and shortened, while
coverage of specific topics has been expanded and updated as
needed. Study is facilitated by modern page design. Inserted
throughout each chapter are more numerous, short paragraphs
that indicate how the information presented can be used
medically and which emphasize the foundational relevance of
the material learned.
The art and other figures are presented in each chapter,
with the goal to simplify learning and integration with
related material. The McGraw-Hill medical illustrations,
now used throughout the text, are the most useful, thorough,
and attractive of any similar medical textbook. Electron and
light micrographs have been replaced throughout the book
as needed, and they again make up a complete atlas of cell,
tissue, and organ structures fully compatible with the students'
own collection of glass or digital slides. Health science
students whose medical library offers AccessMedicine among
its electronic resources (which includes more than 95% of
U.S. medical schools) can access a complete human histology
Laboratory Guide linked to the virtual microscope at the URL
given below. This digital Laboratory Guide, which is new with
this edition of the text and unique among learning resources
offered by any histology text and atlas, provides both links
to the appropriate microscope slides needed for each topic
and links to the correlated figures or tables in the text. Those
without AccessMedicine will lack the digital Laboratory Guide,
but may still study and utilize the 150 virtual microscope
slides of all human tissues and organs, which are available at:
http://medsci.indiana.edu/junqueira/virtual/junqueira.htm.
As with the previous edition, the book facilitates learning
by its organization:
• An opening chapter reviews the histological techniques
that allow understanding of cell and tissue structure.
• Two chapters then summarize the structural and
functional organization of human cell biology,
presenting the cytoplasm and nucleus separately.
• The next seven chapters cover the four basic tissues that
make up our organs: epithelia, connective tissue (and its
major sub-types), nervous tissue, and muscle.
• Remaining chapters explain the organization and
functional significance of these tissues in each of
the body's organ systems, closing with up-to-date
consideration of cells in the eye and ear.
For additional review of what's been learned or to
assist rapid assimilation of the material in Junqueira's Basic
Histology, McGraw-Hill has published a set of 200 full-color
Basic Histology Flash Cards, also authored by Anthony
Mescher. Each card includes images of key structures to
identify, a summary of important facts about those structures,
and a clinical comment. This valuable learning aid is available
as a set of actual cards from Amazon.com, or as an app for
smartphones or tablets from the online App Store.
With its proven strengths and the addition ofnew features,
I am confident that Junqueira's Basic Histology will continue
as one of the most valuable and most widely read educational
resources in histology. Users are invited to provide feedback
to the author with regard to any aspect of the book's features.
Anthony L Mescher
Indiana University School of Medicine
mescher@indiana.edu
vii
This page intentionally left blank
Acknowledgments
I wish to thank the students at Indiana University School of
Medicine and the undergraduates at Indiana University with
whom I have studied histology and cell biology for over 35 years
and from whom I have learned much about presenting basic
concepts most effectively. 'Their input has greatly helped in the
task ofmaintaining and updating the presentations in this classic
textbook. As with the last edition, the help of Sue Childress and
Dr. Mark Braun was invaluable in slide preparation and the
virtual microscope for human histology respectively.
As with the last edition, the present text includes ten
multiple-choice questions at the end of each chapter, aimed
to test the learner's retention and understanding of important
points in that body of material Many of these questions
were used in my courses, but others are taken or modified
from a few of the many excellent review books published by
McGraw-Hill!Lange for students preparing to take the U.S.
Medical Licensing Examination. 'These include Histology and
Cell Biology: Examination and Board Review, by Douglas
Paulsen; USMLE Road Map: Histology, by Harold Sheedlo; and
Anatomy, Histology, & Cell Biology: PreTest Self-Assessment &
Review, by Robert Klein and George Enders. 'The use here
of questions from these valuable resources is gratefully
acknowledged. Students are referred to those review books for
hundreds of additional self-assessment questions.
I am also grateful to my colleagues and reviewers from
throughout the world who provided specialized expertise or
original photographs, which are also acknowledged in figure
captions. I thank those professors and students in the United
States and countries throughout the world who provided useful
suggestions that have improved the new edition of ]unqueira's
Basic Histology. Finally, I am pleased to acknowledge the help
and collegiality provided by the staffofMcGraw-Hill, especially
editors Michael Weitz and Brian Kearns, whose work made
possible publication of this 15th edition of Junqueira's Basic
Histology.
Anthony L. Mescher
Indiana University School of Medicine
mescher@indiana.edu
ix
This page inte7tionally ldt blank
CHAPTER
Histology &Its
Methods of Study
PREPARATION OF TISSUES FOR STUDY 1 AUTORADIOGRAPHY 9
Fixation 1 CELL &TISSUE CULTURE 10
Embedding & Sectioning 3
ENZYME HISTOCHEMISTRY 10
Staining 3
LIGHT MICROSCOPY 4 VISUALIZING SPECIFIC MOLECULES 10
Bright-Field Microscopy 4 Immunohistochemistry 11
Fluorescence Microscopy 5 Hybridization Techniques 12
Phase-Contrast Microscopy 5 INTERPRETATION OF STRUCTURES IN TISSUE
Confocal Microscopy 5 SECTIONS 14
Polarizing Microscopy 7 SUMMARY OF KEY POINTS 15
ELECTRON MICROSCOPY 8 ASSESS YOUR KNOWLEDGE 16
Transmission Electron Microscopy 8
Scanning Electron Microscopy 9

H
istology is the study of the tissues of the body and
how these tissues are arranged to constitute organs.
This subject involves all aspects of tissue biology, with
the focus on how cells' structure and arrangement optimize
functions specific to each organ.
Tissues have two interacting components: cells and extra-
cellular matrix (ECM). The ECM consists of many kinds of
macromolecules, most of which form complex structures,
such as collagen fibrils. The ECM supports the cells and con-
tains the fluid transporting nutrients to the cells, and carry-
ing away their wastes and secretory products. Cells produce
the ECM locally and are in turn strongly influenced by matrix
molecules. Many matrix components bind to specific cell
surface receptors that span the cell membranes and connect
to structural components inside the cells, forming a contin-
uum in which cells and the ECM function together in a well-
coordinated manner.
During development, cells and their associated matrix
become functionally specialized and give rise to fundamen-
tal types of tissues with characteristic structural features.
Organs are formed by an orderly combination of these tissues,
and their precise arrangement allows the functioning of each
organ and of the organism as a whole.
The small size of cells and matrix components makes his-
tology dependent on the use of microscopes and molecular
methods of study. Advances in biochemistry, molecular biol-
ogy, physiology, immunology, and pathology are essential for
a better knowledge of tissue biology. Familiarity with the tools
and methods of any branch of science is essential for a proper
understanding of the subject. This chapter reviews common
methods used to study cells and tissues, focusing on micro-
scopic approaches.
> PREPARATION OF TISSUES
FOR STUDY
The most common procedure used in histologic research is
the preparation of tissue slices or "sections" that can be exam-
ined visually with transmitted light. Because most tissues and
organs are too thick for light to pass through, thin translu-
cent sections are cut from them and placed on glass slides for
microscopic examination of the internal structures.
The ideal microscopic preparation is preserved so that the
tissue on the slide has the same structural features it had in the
body. However, this is often not feasible because the prepara-
tion process can remove cellular lipid, with slight distortions
of cell structure. The basic steps used in tissue preparation for
light microscopy are shown in Figure 1-1.
Fixation
To preserve tissue structure and prevent degradation by
enzymes released from the cells or microorganisms, pieces of

1
2 CHAPTER 1 • Histology & Its Methods of Study
FIGURE 1-1 Sectioning fixed and embedded tissue.
(a) Fixation Dehydration
Most tissues studied histologically are prepared as shown, with
this sequence of steps (a):
• Fixation: Small pieces of tissue are placed in solutions of
chemicals that cross-link proteins and inactivate degradative
enzymes, which preserve cell and tissue structure.
• Dehydration: The tissue is transferred through a series of
increasingly concentrated alcohol solutions, ending in 100%,
which removes all water.
• Clearing: Alcohol is removed in organic solvents in which
both alcohol and paraffin are miscible.
• Infiltration: The tissue is then placed in melted paraffin until it
becomes completely infiltrated with this substance.
• Embedding: The paraffin-infiltrated tissue is placed in a small
mold with melted paraffin and allowed to harden.
• Trimming: The resulting paraffin block is trimmed to expose
the tissue for sectioning (slicing) on a microtome.
organs are placed as soon as possible after removal from the
body in solutions of stabilizing or cross-linking compounds
called fixatives. Because a fixative must fully diffuse through
the tissues to preserve all cells, tissues are usually cut into
small fragments before fixation to facilitate penetration. To
improve cell preservation in large organs, fixatives are often
introduced via blood vessels, with vascular perfusion allowing
fixation rapidly throughout the tissues.
One widely used fixative for light microscopy is forma-
lin, a buffered isotonic solution of 37% formaldehyde. Both
this compound and glutaraldehyde, a fixative used for electron
Clearing Infiltration
- Embedding
Drive wheel
Block holder
Paraffin block
Tissue
Steel knife
Similar steps are used in preparing tissue for transmission elec-
tron microscopy (TEM), except special fixatives and dehydrating
solutions are used with smaller tissue samples and embedding
involves epoxy resins which become harder than paraffin to allow
very thin sectioning.
(b) A microtome is used for sectioning paraffin-embedded tissues
for light microscopy. The trimmed tissue specimen is mounted
in the paraffin block holder, and each turn of the drive wheel by
the histologist advances the holder a controlled distance, gener-
ally from 1 to 10 1-1m. After each forward move, the tissue block
passes over the steel knife edge and a section is cut at a thickness
equal to the distance the block advanced. The paraffin sections
are placed on glass slides and allowed to adhere, deparaffinized,
and stained for light microscope study. ForTEM, sections less than
1 1-1m thick are prepared from resin-embedded cells using an ultra-
microtome with a glass or diamond knife.
microscopy, react with the amine groups (NH2 ) of proteins,
preventing their degradation by common proteases. Glutaral-
dehyde also cross-links adjacent proteins, reinforcing cell and
ECM structures.
Electron microscopy provides much greater magni-
fication and resolution of very small cellular structures,
and fixation must be done very carefully to preserve addi-
tional "ultrastructural" detail. Typically in such studies,
glutaraldehyde-treated tissue is then immersed in buffered
osmium tetroxide, which preserves (and stains) cellular lip-
ids as well as proteins.

Embedding II Sectioning
To permit thin sectioning, fixed tissues are infiltrated and
embedded in a material that imparts a firm consistency.
Embedding materials include paraffin, used routinely for light
microscopy, and plastic resins, which are adapted for both
light and electron microscopy.
Before infiltration with such media, the fixed tissue must
undergo dehydration by having its water extracted gradually
by transfers through a series of increasing ethanol solutions,
ending in 100% ethanol. The ethanol is then replaced by an
organic solvent miscible with both alcohol and the embedding
medium, a step referred to as clearing because infiltration with
the reagents used here gives the tissue a translucent appearance.
The fully cleared tissue is then placed in melted paraffin
in an oven at 52°-60°C, which evaporates the clearing solvent
and promotes infiltration of the tissue with paraffin, and then
embedded by allowing it to harden in a small container of
paraffin at room temperature. Tissues to be embedded with
plastic resin are also dehydrated in ethanol and then infiltrated
with plastic solvents that harden when cross-linking polymer-
izers are added. Plastic embedding avoids the higher tempera-
tures needed with paraffin, which helps avoid tissue distortion.
The hardened block with tissue and surrounding embed-
ding medium is trimmed and placed for sectioning in an
instrument called a microtome (see Figure 1-1). Paraffin sec-
tions are typically cut at 3-10 f1ln thickness for light microscopy,
but electron microscopy requires sections less than I fUll thick.
One micrometer (1 flln) equals 1/1000 of a millimeter (mm)
or 10-6 m. Other spatial units commonly used in microscopy
are the nanometer (1 nm = 0.00lf1ln = 10~ mm =10-9 m) and
angstrom (1 A= 0.1 nm or 10--4flln). The sections are placed on
glass slides and stained for light microscopy or on metal grids
for electron-microscopic staining and examination.
>> MEDICAL APPLICATION
Biopsies are tissue samples removed during surgery or routine
medical procedures. In the operating room, biopsies are fixed
in vials of formalin for processing and microscopic analysis in
a pathology laboratory. If results of such analyses are required
before the medical procedure is completed, for example to
know whether a growth is malignant before the patient is
closed, a much more rapid processing method is used. The
biopsy is rapidly frozen in liquid nitrogen, preserving cell
structures and at the same time making the tissue hard and
ready for sectioning. A microtome called a cryostilt in a cabi-
net at subfreezing temperature is used to section the block
with tissue, and the frozen sections are placed on slides for
rapid staining and microscopic examination by a pathologist.
Freezing of tissues is also effective in histochemical stud-
ies of very sensitive enzymes or small molecules because
freezing, unlike fixation, does not inactivate most enzymes.
Finally, because clearing solvents often dissolve cell lipids in
fixed tissues, frozen sections are also useful when structures
containing lipids are to be studied histologically.
Preparation of Tissues for Study 3
Staining
n
Most cells and extracellular material are completely color-
less, and to be studied microscopically tissue sections must ,.
:I:
be stained (dyed). Methods of staining have been devised that -a
....
make various tissue components not only conspicuous but also m
distinguishable from one another. Dyes stain material more or
less selectively; often behaving like acidic or basic compounds
and forming electrostatic (salt) linkages with ionizable radicals
ofmacromolecules in tissues. Cell components, such as nucleic
..
::a
acids with a net negative charge (anionic), have an affinity for
basic dyes and are termed basophilic; cationic components,
such as proteins with many ionized amino groups, stain more
readily with acidic dyes and are termed acidophilic.
Examples of basic dyes include toluidine blue, aldan blue,
and methylene blue. Hematoxylin behaves like a basic dye,
staining basophilic tissue components. The main tissue com-
ponents that ionize and react with basic dyes do so because of
acids in their composition (DNA, RNA, and glycosaminogly-
cans). Acid dyes (eg, eosin, orange G, and acid fuchsin) stain
the acidophilic components of tissues such as mitochondria,
secretory granules, and collagen. •
Of all staining methods, the simple combination of
hematoxylin and eosin (H&E) is used most commonly.
Hematoxylin stains DNA in the cell nucleus, RNA-rich por-
tions of the cytoplasm, and the matrix of cartilage, produc-
ing a dark blue or purple color. In contrast, eosin stains other
cytoplasmic structures and collagen pink (Figure 1-2a). Here
eosin is considered a counterstain, which is usually a single
dye applied separately to distinguish additional features of a
tissue. More complex procedures, such as trichrome stains (eg,
Masson's trichrome), allow greater distinctions among various
extracellular tissue components.
The periodic acld-Schlff (PAS) reaction utilizes the
hexose rings of polysaccharides and other carbohydrate-rich
tissue structures and stains such macromolecules distinctly
purple or magenta. Figure 1-2b shows an example of cells with
carbohydrate-rich areas well-stained by the PAS reaction. The
DNA of cell nuclei can be specifically stained using a modifica-
tion of the PAS procedure called the Feulgen reaction.
Basophilic or PAS-positive material can be further identi-
fied by enzyme digestion, pretreatment of a tissue section with
an enzyme that specifically digests one substrate. For example,
pretreatment with ribonuclease will greatly reduce cytoplas-
mic basophilia with little overall effect on the nucleus, indicat-
ing the importance of RNA for the cytoplasmic staining.
Lipid-rich structures of cells are revealed by avoiding the
processing steps that remove lipids, such as treatment with
heat and organic solvents, and staining with lipid-soluble
dyes such as Sudan black, which can be useful in diagnosis
of metabolic diseases that involve intracellular accumulations
of cholesterol, phospholipids, or glycolipids. Less common
methods of staining can employ metal impregnation tech-
niques, typically using solutions of silver salts to visual certain
ECM fibers and specific cellular elements in nervous tissue.
The Appendix lists important staining procedures used for
most of the light micrographs in this book.
4 CHAPTER 1 • Histology & Its Methods of Study
FIGURE 1-2 Hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining.
Micrographs of epithelium lining the small intestine, (a) stained
with H&E, and (b) stained with the PAS reaction for glycoproteins.
With H&E, basophilic cell nuclei are stained purple, while cyto-
plasm stains pink. Cell regions with abundant oligosaccharides
on glycoproteins, such as the ends of the cells at the lumen (L)
or the scattered mucus-secreting goblet cells (G), are poorly
stained. With PAS, however, cell staining is most intense at the
Slide preparation, from tissue fixation to observation
with a light microscope, may take from 12 hours to 2~ days,
depending on the size of the tissue, the embedding medium,
and the method of staining. The final step before microscopic
observation is mounting a protective glass coverslip on the
slide with clear adhesive.
> LIGHT MICROSCOPY
Conventional bright-field microscopy and more specialized
applications like fluorescence, phase-contrast, confocal, and
polarizing microscopy are all based on the interaction of light
with tissue components and are used to reveal and study tissue
features.
Bright-Field Microscopy
With the bright-field microscope, stained tissue is examined
with ordinary light passing through the preparation. As shown
in Figure 1-3, the microscope includes an optical system and
mechanisms to move and focus the specimen. The optical
components are the condenser focusing light on the object
to be studied; the objective lens enlarging and projecting the
image of the object toward the observer; and the eyepiece
lumen, where projecting microvilli have a prominent layer of
glycoproteins at the lumen (L) and in the mucin-rich secretory
granules of goblet cells. Cell surface glycoproteins and mucin are
PAS-positive because of their high content of oligosaccharides
and polysaccharides, respectively. The PAS-stained tissue was
counterstained with hematoxylin to show the cell nuclei.
(a. X400; b. X300)
(or ocular lens) further magnifying this image and projecting
it onto the viewer's retina or a charge-coupled device (CCD)
highly sensitive to low light levels with a camera and monitor.
The total magnification is obtained by multiplying the magni-
fying power of the objective and ocular lenses.
The critical factor in obtaining a crisp, detailed image
with a light microscope is its resolving power, defined as the
smallest distance between two structures at which they can be
seen as separate objects. The maximal resolving power of the
light microscope is approximately 0.2 fllll• which can permit
clear images magnified 1000-1500 times. Objects smaller or
thinner than 0.2 fllll (such as a single ribosome or cytoplasmic
microfilament) cannot be distinguished with this instrument.
Likewise, two structures such as mitochondria will be seen as
only one object if they are separated by less than 0.2 fllll· The
microscope's resolving power determines the quality of the
image, its clarity and richness of detail, and depends mainly on
the quality of its objective lens. Magnification is of value only
when accompanied by high resolution. Objective lenses pro-
viding higher magnification are designed to also have higher
resolving power. The eyepiece lens only enlarges the image
obtained by the objective and does not improve resolution.
Virtual microscopy, typically used for study of bright-
field microscopic preparations, involves the conversion of a

FIGURE 1-3 Componentsandlightpathofa
bright-field microscope.
Stand
I
translation
mechanism
Photograph of a bright-field light microscope showing its
mechanical components and the pathway of light from the
substage lamp to the eye of the observer. The optical system
has three sets of lenses:
• The condenser collects and focuses a cone of light that
illuminates the tissue slide on the stage.
• Objective lenses enlarge and project the illuminated
image of the object toward the eyepiece. Interchangeable
objectives with different magnifications routinely used in
histology include X4 for observing a large area (field) of the
tissue at low magnification; Xl 0 for medium magnification
of a smaller field; and X40 for high magnification of more
detailed areas.
• The two eyepieces or oculars magnify this image another
Xl 0 and project it to the viewer, yielding a total magnifica-
tion of X40, Xl 00, or X400.
(Used with permission from Nikon Instruments.)
stained tissue preparation to high-resolution digital images
and permits study of tissues using a computer or other digi-
tal device, without an actual stained slide or a microscope. In
this technique, regions of a glass-mounted specimen are cap-
tured digitally in a grid-like pattern at multiple magnifications
using a specialized slide-scanning microscope and saved as
thousands of consecutive image files. Software then converts
this dataset for storage on a server using a format that allows
access, visualization, and navigation of the original slide with
common web browsers or other devices. With advantages in
cost and ease of use, virtual microscopy is rapidly replacing
light microscopes and collections of glass slides in histology
laboratories for students.
Light Microscopy 5
Fluorescence Microscopy
n
When certain cellular substances are irradiated by light of a :I:
proper wavelength, they emit light with a longer wavelength- >
~
a phenomenon called fluorescence. In fluorescence
-1
microscopy, tissue sections are usually irradiated with ultra- m
violet (UV) light and the emission is in the visible portion of
the spectrum. The fluorescent substances appear bright on
a dark background. For fluorescent microscopy, the instru-
ment has a source of UV or other light and filters that select
..
:u
rays of different wavelengths emitted by the substances to be
visualized.
Fluorescent compounds with affinity for specific cell
macromolecules may be used as fluorescent stains. Acridine
orange, which binds both DNA and RNA, is an example.
When observed in the fluorescence microscope, these nucleic
acids emit slightly different fluorescence, allowing them to be
localized separately in cells (Figure 1-4a). Other compounds,
such as DAPI and Hoechst, stain specifically bind DNA and
are used to stain cell nuclei, emitting a characteristic blue fluo-
rescence under UV. Another important application of fluores-
cence microscopy is achieved by coupling compounds such as •
fluorescein to molecules that will specifically bind to certain I"'"
IS'
cellular components and thus allow the identification of these ....:::r
structures under the microscope (Figure 1-4b). Antibodies s::
;:;·
labeled with fluorescent compounds are extremely important
in immunohistologic staining. (See the section Visualizing a
1ft
Specific Molecules.) s
~
Phase-Contrast Microscopy
Unstained cells and tissue sections, which are usually trans-
parent and colorless, can be studied with these modified
light microscopes. Cellular detail is normally difficult to see
in unstained tissues because all parts of the specimen have
roughly similar optical densities. Phase-contrast micros-
copy, however, uses a lens system that produces visible images
from transparent objects and, importantly; can be used with
living, cultured cells (Figure 1-5).
Phase-contrast microscopy is based on the principle that
light changes its speed when passing through cellular and
extracellular structures with different refractive indices. These
changes are used by the phase-contrast system to cause the
structures to appear lighter or darker in relation to each other.
Because they allow the examination of cells without fixation or
staining, phase-contrast microscopes are prominent tools in
all cell culture laboratories. A modification of phase-contrast
microscopy is differential interference contrast micros-
copy with Nomarski optics, which produces an image of liv-
ing cells with a more apparent three-dimensional (3D) aspect
(Figure 1-Sc).
Confocal Microscopy
With a regular bright-field microscope, the beam of light
is relatively large and fills the specimen. Stray (excess) light
reduces contrast within the image and compromises the
6 CHAPTER 1 • Histology & Its Methods of Study
FIGURE 1-4 Appearance of cells with fluorescent microscopy.
Components of cells are often stained with compounds visible by
fluorescence microscopy.
(a) Acridine orange binds nucleic acids and causes DNA in cell
nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to
appear orange in these cells of a kidney tubule.
(b) Cultured cells stained with DAPI (4',6-diamino-2-phenylindole)
that binds DNA and with fluorescein phalloidin that binds actin
FIGURE 1-5 Unstained cells' appearance in three types of light microscopy.
Living neural crest cells growing in culture appear differently
with various techniques of light microscopy. Here the same field
of unstained cells, including two differentiating pigment cells, is
shown using three different methods (all X200):
(a) Bright-field microscopy: Without fixation and staining, only
the two pigment cells can be seen.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and
cytoplasmic structures with different refractive indices affect
filaments show nuclei with blue fluorescence and actin filaments
stained green. Important information such as the greater density
of microfilaments at the cell periphery is readily apparent. (Both
XSOO)
(Figure 7-4b, used with permission from Drs Claire E. Walczak
and Rania Rizk, Indiana University School of Medicine,
Bloomington.)
in-phase light differently and produce an image of these features
in all the cells.
(c) Differential interference contrast microscopy: Cellular details
are highlighted in a different manner using Nomarski optics.
Phase-contrast microscopy, with or without differential interfer-
ence, is widely used to observe live cells grown in tissue culture.
(Used with permission from Dr Sherry Rogers, Department of Cell
Biology and Physiology, University of New Mexico, Albuquerque, NM.)
 Light Microscopy 7

such optical sections at a series of focal planes through the

FIGURE 1-6 Principle of confocal microscopy. n
specimen allows them to be digitally reconstructed into a
:I:
3D image.
>
~

Polarizing Microscopy -1
m

..
Polarizing microscopy allows the recognition of stained or :u
unstained structures made of highly organized subunits.
When normal light passes through a polarizing filter, it exits
vibrating in only one direction. If a second filter is placed in
the microscope above the first one, with its main axis per-
Detector
I pendicular to the first filter, no light passes through. If, how-
ever, tissue structures containing oriented macromolecules
are located between the two polarizing filters, their repeti-
tive structure rotates the axis of the light emerging from the
polarizer and they appear as bright structures against a dark
background (Figure 1-7). The ability to rotate the direction
of vibration of polarized light is called birefringence and is

--Beam
splitter FIGURE 1-7 Tissue appearance with bright-field
and polarizing microscopy.
•
I"'"
IS'
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i:
;:;·
a
1ft

s
~

Although a very small spot of light originating from one plane

ofthe section crosses the pinhole and reaches the detector,
rays originating from other planes are blocked by the blind.
Thus, only one very thin plane of the specimen is focused at a
time. The diagram shows the practical arrangement of a confo-
cal microscope. Light from a laser source hits the specimen and
is reflected. A beam splitter directs the reflected light to a pin-
hole and a detector. Light from components of the specimen
that are above or below the focused plane is blocked by the
blind. The laser scans the specimen so that a larger area of the
specimen can be observed.

resolving power of the objective lens. Confocal microscopy

(Figure 1-6) avoids these problems and achieves high reso-
lution and sharp focus by using (1) a small point of high-
intensity light, often from a laser and (2) a plate with a
pinhole aperture in front of the image detector. The point
light source, the focal point of the lens, and the detector's
pinpoint aperture are all optically conjugated or aligned to
Polarizing light microscopy produces an image only of material
each other in the focal plane (confocal), and unfocused light having repetitive, periodic macromolecular structure; features
does not pass through the pinhole. This greatly improves without such structure are not seen. Pieces of thin, unsec-
resolution of the object in focus and allows the localization tioned mesentery were stained with red picrosirius, orcein, and
of specimen components with much greater precision than hematoxylin, placed on slides and observed by bright-field (a)
and polarizing (b) microscopy.
with the bright-field microscope.
Confocal microscopes include a computer-driven mirror (a) With bright-field microscopy, collagen fibers appear red,
with thin elastic fibers and cell nuclei darker. (X40)
system (the beam splitter) to move the point of illumination
(b) With polarizing microscopy, only the collagen fibers are
across the specimen automatically and rapidly. Digital images
visible and these exhibit intense yellow or orange birefrin-
captured at many individual spots in a very thin plane of focus gence. (a: X40; b: X1 00)
are used to produce an "optical section'' of that plane. Creating
8 CHAPTER 1 • Histology & Its Methods of Study
a feature of crystalline substances or substances containing
highly oriented molecules, such as cellulose, collagen, micro-
tubules, and actin filaments.
The utility of all light microscopic methods is greatly
extended through the use of digital cameras. Many features
of digitized histologic images can be analyzed quantitatively
using appropriate software. Such images can also be enhanced
to allow objects not directly visible through the eyepieces to be
examined on a monitor.
> ELECTRON MICROSCOPY
Transmission and scanning electron microscopes are based on
the interaction of tissue components with beams of electrons.
FIGURE 1-8 Electron microscopes.
Intermediate lens--~
Projector lens----~
Image on viewing - -----4!;
screen
Electron detector
with ceo camera
(a) Transmission electron microscope
Electron microscopes are large instruments generally housed in a
specialized EM facility.
(a) Schematic view of the major components of a transmission elec-
tron microscope (TEM), which is configured rather like an upside-
down light microscope. With the microscope column in a vacuum, a
metallic (usually tungsten) filament (cathode) at the top emits elec-
trons that travel to an anode with an accelerating voltage between
60 and 120 kV. Electrons passing through a hole in the anode form
a beam that is focused electromagnetically by circular electric
coils in a manner analogous to the effect of optical lenses on light.
The first lens is a condenser focusing the beam on the section.
Some electrons interact with atoms in the section, being absorbed
or scattered to different extents, while others are simply transmit-
ted through the specimen with no interaction. Electrons reaching
the objective lens form an image that is then magnified and finally
projected on a fluorescent screen or a charge-coupled device
(CCD) monitor and camera.
The wavelength in an electron beam is much shorter than that
of light, allowing a 1000-fold increase in resolution.
Transmission Electron Microscopy
The transmission electron microscope (TEM) is an imag-
ing system that permits resolution around 3 nm. This high
resolution allows isolated particles magnified as much as
400,000 times to be viewed in detail. Very thin (40-90 nm),
resin-embedded tissue sections are typically studied by TEM
at magnifications up to approximately 120,000 times.
Figure 1-8a indicates the components of a TEM and the
basic principles of its operation: a beam of electrons focused
using electromagnetic "lenses" passes through the tissue sec-
tion to produce an image with black, white, and intermediate
Cathode------~
(b) Scanning electron microscope
In a TEM image areas of the specimen through which electrons
passed appear bright (electron lucent), while denser areas or
those that bind heavy metal ions during specimen preparation
absorb or deflect electrons and appear darker (electron dense).
Such images are therefore always black, white, and shades of gray.
(b) The scanning electron microscope (SEM) has many similarities
to a TEM. However, here the focused electron beam does not pass
through the specimen, but rather is moved sequentially (scanned)
from point to point across its surface similar to the way an electron
beam is scanned across a television tube or screen. For SEM speci-
mens are coated with metal atoms with which the electron beam
interacts, producing reflected electrons and newly emitted secondary
electrons. All of these are captured by a detector and transmitted to
amplifiers and processed to produce a black-and-white image on the
monitor. The SEM shows only surface views of the coated specimen
but with a striking 30, shadowed quality. The inside of organs or cells
can be analyzed after sectioning to expose their internal surfaces.

shades of gray regions. These regions of an electron micro-
graph correspond to tissue areas through which electrons
passed readily (appearing brighter or electron-lucent) and
areas where electrons were absorbed or deflected (appearing
darker or more electron-dense). To improve contrast and reso-
lution in TEM, compounds with heavy metal ions are often
added to the fixative or dehydrating solutions used for tissue
preparation. These include osmium tetroxide, lead citrate,
and uranyl compounds, which bind cellular macromolecules,
increasing their electron density and visibility.
Cryofracture and freeze etching are techniques that
allow TEM study of cells without fixation or embedding and
have been particularly useful in the study of membrane struc-
ture. In these methods, very small tissue specimens are rap-
idly frozen in liquid nitrogen and then cut or fractured with a
knife. A replica of the frozen exposed surface is produced in a
vacuum by applying thin coats of vaporized platinum or other
metal atoms. After removal of the organic material, the replica
of the cut surface can be examined by TEM. With membranes
the random fracture planes often split the lipid bilayers, expos-
ing protein components whose size, shape, and distribution
are difficult to study by other methods.
Scanning Electron Microscopy
Scanning electron microscopy (SEM) provides a high-
resolution view of the surfaces of cells, tissues, and organs. Like
the TEM, this microscope produces and focuses a very narrow
beam of electrons, but in this instrument the beam does not
pass through the specimen (Figure 1-8b). Instead, the surface
of the specimen is first dried and spray-coated with a very thin
FIGURE 1-9 Microscopicautoradiography.
Auto radiographs are tissue preparations in which particles called
silver grains indicate the cells or regions of cells in which specific
macromolecules were synthesized just prior to fixation. Shown
here are autoradiographs from the salivary gland of a mouse
injected with 3H-fucose 8 hours before tissue fixation. Fucose was
incorporated into oligosaccharides, and the free 3 H-fucose was
removed during fixation and sectioning of the gland. Autoradio-
graphic processing and microscopy reveal locations of newly syn-
thesized glycoproteins containing that sugar.
Autoradiography 9
layer of heavy metal (often gold) that reflects electrons in a
beam scanning the specimen. The reflected electrons are cap- n
:I:
tured by a detector, producing signals that are processed to pro-
>
duce a black-and-white image. SEM images are usually easy to ~
interpret because they present a three-dimensional view that -1
m
appears to be illuminated in the same way that large objects are
seen with highlights and shadows caused by light.
>AUTORADIOGRAPHY
..
:u
Microscopic autoradiography is a method of localizing
newly synthesized macromolecules in cells or tissue sections.
Radioactively labeled metabolites (nucleotides, amino acids,
sugars) provided to the living cells are incorporated into spe-
cific macromolecules (DNA, RNA, protein, glycoproteins, and
polysaccharides) and emit weak radiation that is restricted
to those regions where the molecules are located. Slides with
radiolabeled cells or tissue sections are coated in a darkroom
with photographic emulsion in which silver bromide crystals
act as microdetectors of the radiation in the same way that
they respond to light in photographic film. After an adequate
,.c•
exposure time in lightproof boxes, the slides are developed
photographically. Silver bromide crystals reduced by the radia- ..
0
ii'l
tion produce small black grains of metallic silver, which under a.
either the light microscope or TEM indicate the locations of ~·
radiolabeled macromolecules in the tissue (Figure 1-9). ii'l
Much histological information becomes available by 'a
:::r
autoradiography. If a radioactive precursor of DNA (such "<
as tritium-labeled thymidine) is used, it is possible to know
which cells in a tissue (and how many) are replicating DNA
(a) Black grains of silver from the light-sensitive material coating
the specimen are visible over cell regions with secretory granules
and the duct indicating glycoprotein locations. (XlSOO)
(b) The same tissue prepared for TEM autoradiography shows sil-
ver grains with a coiled or amorphous appearance again localized
mainly over the granules (G) and in the gland lumen (L). (X7500)
(Figure 7-9b, used with permission from Drs Ticiano G. Lima and
A. Antonio Haddad, School of Medicine, Ribeircio Preto, Brazil.)
10 CHAPTER 1 • Histology & Its Methods of Study
and preparing to divide. Dynamic events may also be analyzed.
For example, if one wishes to know where in the cell protein is
produced, if it is secreted, and its path in the cell before being
secreted, several animals are injected with a radioactive amino
acid and tissues collected at different times after the injections.
Autoradiography of the tissues from the sequential times will
indicate the migration of the radioactive proteins.
> CELL &TISSUE CULTURE
Live cells and tissues can be maintained and studied outside
the body in culture (in vitro). In the organism (in vivo), cells
are bathed in fluid derived from blood plasma and containing
many different molecules required for survival and growth.
Cell culture allows the direct observation of cellular behavior
under a phase-contrast microscope, and many experiments
technically impossible to perform in the intact animal can be
accomplished in vitro.
The cells and tissues are grown in complex solutions of
known composition (salts, amino acids, vitamins) to which
serum or specific growth factors are added. Cells to be cultured
are dispersed mechanically or enzymatically from a tissue or
organ and placed with sterile procedures in a clear dish to
which they adhere, usually as a single layer (see Figure 1-5).
Such preparations are called primary cell cultures. Some
cells can be maintained in vitro for long periods because they
become immortalized and constitute a permanent cell line.
Most cells obtained from normal tissues have a finite, geneti-
cally programmed life span. However, certain changes (some
related to oncogenes; see Chapter 3) can promote cell immor-
tality, a process called transformation, and are similar to
the initial changes in a normal cell's becoming a cancer cell
Improvements in culture technology and use of specific growth
factors now allow most cell types to be maintained in vitro.
As shown in Chapter 2, incubation of living cells in vitro
with a variety of new fluorescent compounds that are seques-
tered and metabolized in specific compartments of the cell
provides a new approach to understanding these compart-
ments both structurally and physiologically. Other histologic
techniques applied to cultured cells have been particularly
important for understanding the locations and functions of
microtubules, microfilaments, and other components of the
cytoskeleton.
>> MEDICAL APPLICATION
Cell culture is very widely used to study molecular changes
that occur in cancer; to analyze infectious viruses, myco-
plasma, and some protozoa; and for many routine genetic or
chromosomal analyses. Cervical cancer cells from a patient
later identified as Henrietta Lacks, who died from the disease
in 1951, were used to establish one of the ti rst cell lines,
called HeLa cells, which are still used in research on cellular
structure and function throughout the world.
> ENZYME HISTOCHEMISTRY
Enzyme histochemistry (or cytochemistry) is a method for
localizing cellular structures using a specific enzymatic activ-
ity present in those structures. To preserve the endogenous
enzymes, histochemical procedures usually use unfixed or
mildly fixed tissue, which is sectioned on a cryostat to avoid
adverse effects of heat and organic solvents on enzymatic
activity. For enzyme histochemistry: (1) tissue sections are
immersed in a solution containing the substrate of the enzyme
to be localized; (2) the enzyme is allowed to act on its sub-
strate; (3) the section is then put in contact with a marker
compound that reacts with a product of the enzymatic action
on the substrate; and (4) the final product from the marker,
which must be insoluble and visible by light or electron
microscopy, precipitates over the site of the enzymes, identify-
ing their location.
Examples of enzymes that can be detected histochemi-
cally include the following:
• Phosphatases, which remove phosphate groups from
macromolecules (Figure 1-10).
• Dehydrogenases, which transfer hydrogen ions from
one substrate to another, such as many enzymes of the
citric acid (Krebs) cycle, allowing histochemical identifi-
cation of such enzymes in mitochondria.
• Peroxidase, which promotes the oxidation of sub-
strates with the transfer of hydrogen ions to hydrogen
peroxide.
>> MEDICAL APPLICATION
Many enzyme histochemical procedures are used in the
medical laboratory, including Perls'Prussian blue reaction for
Iron (used to diagnose the Iron storage diseases, hemochro-
matosis and hemosiderosis), the PAS-amylase and aldan blue
reactions for polysaccharides (to detect glycogenosis and
mucopolysaccharidosls), and reactions for lipids and sphin-
golipids (to detect sphingolipidosis).
> VISUALIZING SPECIFIC MOLECULES
A specific macromolecule present in a tissue section may also
be identified by using tagged compounds or macromolecules
that bind specifically with the molecule of interest. The com-
pounds that interact with the molecule must be visible with
the light or electron microscope, often by being tagged with a
detectible label. The most commonly used labels are fluores-
cent compounds, radioactive atoms that can be detected with
autoradiography, molecules of peroxidase or other enzymes
that can be detected with histochemistry, and metal (usually
gold) particles that can be seen with light and electron micros-
copy. These methods can be used to detect and localize specific
sugars, proteins, and nucleic acids.

FIGURE 1-10 Enzyme histochemistry.
(a) Micrograph of cross sections of kidney tubules treated
histochemically to demonstrate alkaline phosphatases (with
maximum activity at an alkaline pH) showing strong activity of
this enzyme at the apical surfaces of the cells at the lumens (L)
of the tubules. (X200)
(b) TEM image of a kidney cell in which acid phosphatase has
been localized histochemically in three lysosomes (Ly) near the
nucleus (N). The dark material within these structures is lead
phosphate that precipitated in places with acid phosphatase
activity. (X25,000)
(Figure 7-7 Ob, used with permission from Dr Eduardo
Katchburian, Department of Morphology, Federal University of
Sao Paulo, Brazil.)
Visualizing Specific Molecules 11
Examples of molecules that interact specifically with
n
other molecules include the following:
:I:
• Phalloidin, a compound extracted from mushroom, >
~
Amanita phalloides, interacts strongly with the actin pro-
-1
tein of microfilaments. m
• Protein A, purified from Staphylococcus aureus bacte-
ria, binds to the Fe region of antibody molecules, and
can therefore be used to localize naturally occurring or
applied antibodies bound to cell structures.
..
:u
• Lectins, glycoproteins derived mainly from plant seeds,
bind to carbohydrates with high affinity and specificity.
Different lectins bind to specific sugars or sequences of
sugar residues, allowing fluorescently labeled lectins to
be used to stain specific glycoproteins or other macro-
molecules bearing specific sequences of sugar residues.
Immunohistochemistry
A highly specific interaction between macromolecules is that
between an antigen and its antibody. For this reason, labeled
antibodies are routinely used in immunohistochemistry
to identify and localize many specific proteins, not just •
those with enzymatic activity that can be demonstrated by s
1ft
c
histochemistry. !.
The body's immune cells interact with and produce anti- iii'
:;·
bodies against other macromolecules-called antigens-that 1&2
are recognized as "foreign;' not a normal part of the organism,
and potentially dangerous. Antibodies belong to the immu-
"'n
'a
ID
noglobulin family of glycoproteins and are secreted by lym- 3i
n
phocytes. These molecules normally bind specifically to their s::
0
provoking antigens and help eliminate them.
Widely applied for both research and diagnostic pur-
ifc
poses, every immunohistochemical technique requires an iD
1ft
antibody against the protein that is to be detected. This means
that the protein must have been previously purified using bio-
chemical or molecular methods so that antibodies against it
can be produced. To produce antibodies against protein x of a
certain animal species (eg, a human or rat), the isolated pro-
tein is injected into an animal of another species (eg, a rabbit
or a goat). If the protein's amino acid sequence is sufficiently
different for this animal to recognize it as foreign-that is, as
an antigen-the animal will produce antibodies against the
protein.
Different groups (clones) of lymphocytes in the injected
animal recognize different parts of protein x and each clone
produces an antibody against that part. These antibodies are
collected from the animal's plasma and constitute a mixture
of polyclonal antibodies, each capable ofbinding a different
region of protein x.
It is also possible, however, to inject protein x into a
mouse and a few days later isolate the activated lymphocytes
and place them into culture. Growth and activity of these cells
can be prolonged indefinitely by fusing them with lymphocytic
tumor cells to produce hybridoma cells. Different hybridoma
clones produce different antibodies against the several parts of
12 CHAPTER 1 • Histology & Its Methods of Study
protein x, and each clone can be isolated and cultured sepa-
rately so that the different antibodies against protein x can be
collected separately. Each of these antibodies is a monoclo-
nal antibody. An advantage to using a monoclonal antibody
rather than polyclonal antibodies is that it can be selected to
be highly specific and to bind strongly to the protein to be
detected, with less nonspecific binding to other proteins that
are similar to the one of interest.
In immunohistochemistry, a tissue section that one
believes contains the protein of interest is incubated in a solu-
tion containing antibody (either monoclonal or polyclonal)
against this protein. The antibody binds specifically to the
protein and after a rinse the protein's location in the tissue or
cells can be seen with either the light or electron microscope
by visualizing the antibody. Antibodies are commonly tagged
with fluorescent compounds, with peroxidase or alkaline
phosphatase for histochemical detection, or with electron-
dense gold particles for TEM.
As Figure 1-11 indicates, there are direct and indirect
methods of immunocytochemistry. The direct method just
involves a labeled antibody that binds the protein of interest.
Indirect immunohistochemistry involves sequential
application of two antibodies and additional washing steps. The
(primary) antibody specifically binding the protein of interest
is not labeled. The detectible tag is conjugated to a second-
ary antibody made in an animal species different ("foreign'')
from that which made the primary antibody. For example, pri-
mary antibodies made by mouse lymphocytes (such as most
monoclonal antibodies) are specifically recognized and bound
by antibodies made in a rabbit or goat injected with mouse
antibody immunoglobulin.
FIGURE 1-11 lmmunocytochemistrytechniques.
-Glass slide--
Direct
Immunocytochemistry (or immunohistochemistry) can be direct
or indirect. Direct immunocytochemistry (left) uses an antibody
made against the tissue protein of interest and tagged directly
with a label such as a fluorescent compound or peroxidase. When
placed with the tissue section on a slide, these labeled antibod-
ies bind specifically to the protein (antigen) against which they
were produced and can be visualized by the appropriate method.
Indirect immunocytochemistry (right) uses first a primary
antibody made against the protein (antigen) of interest and
applied to the tissue section to bind its specific antigen. Then a
The indirect method is used more widely in research and
pathologic tests because it is more sensitive, with the extra
level of antibody binding serving to amplify the visible signal.
Moreover, the same preparation oflabeled secondary antibody
can be used in studies with different primary antibodies (spe-
cific for different antigens) as long as all these are made in the
same species. There are other indirect methods that involve the
use of other intermediate molecules, such as the biotin-avidin
technique, which are also used to amplify detection signals.
Examples of indirect immunocytochemistry are shown in
Figure 1-12, demonstrating the use of this method with cells
in culture or after tissue sectioning for both light microscopy
andTEM.
>> MEDICAL APPLICATION
Because cells in some diseases, including many cancer cells,
often produce proteins unique to their pathologic condition,
immunohistochemistry can be used by pathologists to diag-
nose many diseases, including certain types of tumors and
some virus-infected cells. Table 1-1 shows some applications
of immunocytochemistry routinely used in clinical practice.
Hybridization Techniques
Hybridization usually implies the specific binding between
two single strands of nucleic acid, which occurs under appro-
priate conditions if the strands are complementary. The greater
the similarities of their nucleotide sequences, the more read-
ily the complementary strands form "hybrid" double-strand
molecules. Hybridization at stringent conditions allows the
Labeled
secondary
antibody
Indirect
labeled secondary antibody is obtained that was (1) made in
another species against immunoglobulin proteins (antibodies)
from the species in which the primary antibodies were made and
(2) labeled with a fluorescent compound or peroxidase. When
the labeled secondary antibody is applied to the tissue section, it
specifically binds the primary antibodies, indirectly labeling the
protein of interest on the slide. Because more than one labeled
secondary antibody can bind each primary antibody molecule,
labeling of the protein of interest is amplified by the indirect
method.
 Visualizing Specific Molecules 13

FIGURE 1-12 Cells and tissues stained by immunohistochemistry. n

:I:
>
~

-1
m

..
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the cytoplasm. Primary antibodies against the filament pro-

tein desmin and fluorescein isothiocyanate (FITC)-Iabeled
secondary antibodies were used in the indirect staining
technique, with the nucleus counterstained blue with DAPI.
(X650) •
(b) A section of small intestine treated with an antibody against
s
1ft

the enzyme lysozyme. The secondary antibody labeled with

c
!.
peroxidase was then applied and the localized brown color iii'
produced histochemically with the peroxidase substrate :;·
1&2
3,3'-diamino-azobenzidine (DAB). The method demonstrates
lysozyme-containing structures in scattered macrophages and
in the large clusters of cells. Nuclei were counterstained with
"'n
'a
ID

hematoxylin. (Xl 00) 3i

b
Immunocytochemical methods to localize specific proteins can
be applied to either light microscopic orTEM preparations using
a variety of labels.
(a) A single cultured uterine cell stained fluorescently to reveal
a meshwork of intermediate filaments (green) throughout
n
(c) A section of pancreatic cells in a TEM preparation incubated s::
0
with an antibody against the enzyme amylase and then with
protein A coupled with gold particles. Protein A has high affin- ifc
ity toward antibody molecules and the resulting image reveals iD
1ft
the presence of amylase with the gold particles localized as very
small black dots over dense secretory granules and developing
granules (left). With specificity for immunoglobulin molecules,
labeled protein A can be used to localize any primary antibody.
(XSOOO)
(Figure 7-7 2c, used with permission from Dr Moise Bendayan,
Departments of Pathology and Cell Biology, University of Montreal,
Montreal, Canada.)

TABLE 1-1 Examples of specific antigens with diagnostic importance.

Antigens Diagnosis

Specific cytokeratins Tumors of epithelial origin

Protein and polypeptide hormones Certain endocrine tumors

Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract and breast

Steroid hormone receptors Breast duct cell tumors

Antigens produced by viruses Specific virus infections
Another random document with
no related content on Scribd:
gave from his study upon the garden to the rear; (3) There was a
little hand-cart for luggage in a shed in this garden, which cart
offered itself apt to a dual purpose—(A) to convey down to the shore
a pick and a shovel, together with Uncle Jenico’s colossal wrench,
which, under pretence of its being submitted to some test, had
already been brought to the rectory; (B) to serve as vehicle for the
carrying back of the treasure.
On the top of all which, I ask you, was Mr. Sant the incredulous
humourist he professed to be?
Whatever he thought, however, Uncle Jenico was patently and
irresistibly the enthusiast of the undertaking. He stumped along, dear
soul, his face one moon of hilarity. The adventure was to his very
heart. To be called upon, in such an enterprise, to advertise the
merits of such an invention, his own! It was unspeakable—beyond
expectation! He laughed constantly, holding my arm, and rebuking
me for being a sluggard when I tried to regulate his pace lest he
upset himself.
Harry trundled the cart, making the softest track he could manage,
under the hill towards the Gap. It was a brilliant moonlit night, with a
singing wind. We had brought lanterns; but had no need of them. It
was near as bright as day, indeed, and we sped rapidly on our
course, never having need to pause or pick our way till we reached
the sands. The great shaft of the well, when we stood over against it,
seemed to topple towards us, tragically anticipating its doom. The
sight of it, so lonely and so ancient in this moon-drowned solitude,
thrilled me with a sort of pity. It had stood so long, baffling the winds
and tides, foregathering with such generations of dead and departed
ghosts! And now at last man’s cupidity was scheming to compass
the final ruin of what Nature had been impotent to wreck. Ah! a more
fatal force than any storm! the one against which no monument,
however venerable, is proof.
If the others were touched by this spirit of regret, they were
sensible enough to subordinate it to the inevitably practical. While I
was, literally, mooning, they had already lifted the wrench from the
barrow, and were busy, under Uncle Jenico’s directions, getting it
into position on the sand.
 I can only hastily elucidate the idea of this machine. Pinned to a
sort of frame, or trestle, which was anchored all round with stout
grapnels, and shored up in front against a bracket, was a ship’s
steering-wheel, which the inventor had picked up cheap at a marine
auction. A good rope (length indefinite), to be passed round the
subject of the proposed haulage, and its two ends then carried to the
wheel and clamped, one on each side, to its rim, completed the
design. So disposed, nothing remained but to turn the wheel by its
spokes, when the rope would garrotte the object, and, mechanically
contracting of itself, induce a forward strain.
Now, I know little about scientific values; but certainly in this case
the result justified the means, as you shall hear.
We had got all in place but the rope; and then suddenly Mr. Sant
drew himself up, scratching his head in an unclerical manner.
“Whereabouts is it to be passed round?” he said.
“O!” answered Uncle Jenico: “as high up on the shaft as one can
reach.”
“My good man,” cried the rector sarcastically, “do you really
imagine we are going to haul that thing over by tugging at its base,
or near it?”
“It is tottering already. It is laid bare to its lowest course. These
boys examined and proved it!” answered Uncle Jenico.
Nevertheless, I could see he was taken by surprise and dismayed.
“That may be,” said Mr. Sant, “but——”
He paused, shrugged his shoulders, and laughed out comically.
“O, it will never do!” he said. “We must give this insanity a better
chance. By hook or by crook, we must get the thing fixed up near the
top.”
I started forward.
“I’ll carry the rope up, sir. I know the way. Harry and I climbed it
once before.”
“No,” cried Uncle Jenico, sharply and decisively. “I won’t have you
go on any account, Richard!”
“Then it’s to be me!” cried Harry; and, as I muttered discontentedly,
trying to block his way, he evaded me and ran for the shaft. Mr. Sant,
trailing the rope, followed him, and in a moment they were under its
shadow.
 I chafed, watching them: but my relative was inexorable. And,
indeed, to speak truth, there was considerably more risk in the
venture than formerly before the storm. Harry, however,
accomplished his part in safety; and, while he still dwelt aloft, holding
the loop in place, Mr. Sant captured the two ends of the rope, and
came running towards us with them. In a moment we had pulled
them taut and clamped them in place to the wheel. And then we
hailed Harry to come down, which he did, rather with a run, so afraid
was he of missing any detail of the sport.
Uncle Jenico had already given a half-turn to the wheel, in order to
clinch the hold of the rope; and now he stood in a tense eagerness,
dwelling on the psychologic moment. He held, by right of patent, the
larboard spokes; Mr. Sant, the port. The dear old man was so
wrought up out of feebleness, that I was apprehensive of the part he
insisted upon taking in the manipulation of his own design. He would
not be denied, however; and who could have had the heart to
disappoint him? Was not this the very first time that his genius for
invention promised him a harvest of gold? He took a long breath,
and tightened his hold on the spokes.
Joshua stood rigid, awaiting the result. Harry and I shook on wires,
staring from the wrench to the shaft, and hardly stifling the
exclamations that rose to our lips. It was a solemn moment.
“Go!” cried Uncle Jenico; and the wheel spun a little, stiffened, and
began to cry ominously.
Something cracked; thank Heaven it was only Uncle Jenico’s
braces! The old man tugged and puffed, wrestling with his task.
Suddenly he staggered—the wheel seemed to give and spin away
from him—and he was almost on his face. In the same moment I
fancied the shadow of a night-bird had crossed my vision—and I
looked; and where had been the well was nothing. It was fallen
prone upon the sand, so wearily, so softly, that in that humming wind
no sound of the concussion had reached us.
Hardly suppressing a cry of triumph, we dropped everything, and
raced for the place. The shaft in falling had broken into three pieces,
of which the middle one was in a proportion of two-fourths. The
fracture nearest the base was only three or so inches in width; but
the top fragment was quite detached, and tilted over a little away
from the neck.
Where the shaft had stood was surprisingly little scar in the ground
—nothing to see, in fact, but a pyramid of sand, which had run from
the stuffed base of the well in its parting. Upon this we flung
ourselves, scrambling and scraping like children about a burst sugar
cask. We clawed, as badgers claw, throwing the draff behind us. A
hole opened under our furious assault, and sunk, and deepened—
and revealed nothing. We ran for the tools, and picked and dug like
madmen. Presently Mr. Sant threw down his shovel.
“We are feet below the well bottom. Are you satisfied at last, Mr.
Pilbrow?” he said, really in a quite quarrelsome way. He had been
cheated, he felt, of the fruits of his own condescension.
“No,” snarled Joshua, “I’m not. Here was mud, perhaps, once. It
was a loaded box of iron—we know that. It may have sunk far.”
Mr. Sant laughed offensively. The best of us bear awakening from
engaging dreams badly. As for me, I had desisted from working
when he did, and was sitting disconsolately on the lower part of the
shaft, fumbling with my fingers in the fracture.
All in a moment the blood seemed to rush to my heart, making me
gasp. I jumped to my feet.
“Here it is!” I screeched. “I’ve found it! I felt it!”
My fingers, burrowing through the crack into a choke of sand, had
touched upon the iron-bound corner of a box.
They were all up and swarming about me directly. One by one,
quite cavalier to each other in their eagerness to dive and feel, they
exclaimed and fell back, Some people say that colours are
indiscernible by moonlight. I can answer for the flush which suffused
our rector’s cheek as he looked at Joshua.
But it was Uncle Jenico who commanded the situation.
“We must rope this lowest piece, and pull it away from the other,”
he cried, full of bustle and excitement. “What a providential thought
was this wrench of mine! Hey, my boys? Ha-ha!”
It was brilliantly the obvious course, and at the word we were all
scurrying to put it into execution, Uncle Jenico directing us in a
perfect and quite lovable rapture of self-importance. He and I, when
the rope had been readjusted to its new position, hurried to
manipulate the machine, while the others remained to watch the
result of our efforts on the huge pipe of masonry. We seized the
spokes.
“Right!” said my uncle, with a laugh of joyous confidence.
Now, I don’t know if the first test had amounted to no more than a
little soft extra persuasion applied to an already tottering article. I
know only that that success was not to be repeated.
“Right!” said Uncle Jenico; and the wheel turned under our hands,
tightened, and began to scream as before, only infinitely more
distressfully. We strained our mightiest, putting our backs into it.
“It gives, I think,” said Uncle Jenico, in a suffocating voice.
And with the word, an explosive lash whistled by my ear, the
machine bounded and pitched, and there were we rolling on the
sand amidst a mad wreck of everything.
We were neither of us hurt. Uncle Jenico sat up ruefully. Mr. Sant
came running to us across the sand.
“Anybody killed?” he panted, as he rushed up.
Nobody, by God’s mercy! It was the nearest shave. If I had had a
whisker, it would have been shorn off, I think. The rope had snapped
like a piece of string, and we were right in the path of its recoil.
“Anyhow, I suppose we moved the thing a little?” said Uncle
Jenico.
“Not an inch,” was the answer.
“Eh!” cried my uncle. “I can’t understand. It must have severed
itself on a sharp stone, I suppose.”
“That was the case, without doubt,” said the clergyman, kindly.
“Well, there’s nothing for us now but to take pick and shovel, and dig
out the pith of the thing. It will take a little longer, that’s all.”
Indeed, we found the other two, once assured of our safety,
already hard at the job. It proved a tough one, for the silt inside from
long pressure was grown as compact as mortar, and every fragment
of it had to be chipped off and pulled away—a difficult matter, when
from the depth of our boring it was no longer possible to wield the
pick. However, we got through it, taking turns at the tools, and
working now by lantern light, for the end of the great trunk was
turned from the face of the moon.
 Suddenly Harry, when he and I were once more hammering and
shovelling together, uttered a stifled sound, and scrambled up, so
quickly as half to fracture his skull against the roof of the tube. Then,
holding his head, and squatting out backwards, he gingerly raked
after him a little white thing—a human bone.
I scuttled to join him, and we all looked at one another.
“We’re coming to it,” muttered Mr. Sant; and almost on the instant,
as we plunged in again to resume our burrowing, the end was
wrought. A slab of concreted stuff, falling detached to our renewed
blows and tilting outwards, let down an avalanche of loosened sand,
and, slipping on its torrent—what?
We did not wait to discriminate. The dead, it seemed to us only,
had come sliding and chuckling to meet us half way, with his, “Here
we are again!” like a clown.
“It’s there!” gasped Harry, as we stood up outside. “Some one else
must fetch it—not me: I won’t.”
Joshua dived on the instant: we heard him scuffling and chattering
inside. And then he emerged.
“The rope!” he cried like a madman. “Fetch it—a bit of it—
anything!”
I ran off, unknotted the shorter length from the wreck of the
machine, and returned with it to him. He disappeared again into the
tunnel, drawing the slack after him, and in a minute reissued,
unkempt and agitated beyond measure, and disposed us all to haul.
Without a question we obeyed, and, at his word, set our shoulders to
a simultaneous tug. Slowly the capture responded to our efforts, and
drew out heavily into the open—a great iron-ribbed box, with the
upper half of a human skeleton chained to it by the neck.
Joshua seized the pick, and, before Mr. Sant could stop him, had
parted at a blow the skull from its vertebræ. It leapt and settled,
grinning up at us from the sand.
“That was basely done,” said our rector. “Take your spoil, sir.
These poor remains are my concern.”
Joshua had thrown away the tool, and was standing, as if petrified,
looking down on the chest. It might have measured a yard by two
feet, and some two feet and a half in depth. The wood, under the
corroded clamps of iron, was spongey, half-eaten by water, and, half-
eaten, preserved in sand. But of the immense antiquity of the whole
there was no question.
“We must secure what of these bones we can,” said Mr. Sant.
“Well, Dick? Well, Harry?”
His quiet appeal overcame our repugnance. Once more we
grovelled and groped in the bowels of the well. It was a gruesome
task; but we fulfilled it. Excitement, no doubt—an eagerness to be
done with it, and so earn the sweeter reward of adventure,
stimulated us. At the end we had found, and gathered into a heap
outside, all evidence that remained to mortality of that ancient deed
of murder. It made one’s brain swim to look down on this wonderful
tragic salvage of the centuries. It was all true, then—all true! And
Destiny had made us her instruments in this unspeakable
resurrection!
All this time Joshua, and even my uncle, had remained as if
tranced. Now, suddenly, the former raised his voice in a shrill ecstatic
cry.
“Poor Abel! poor fool! Come, let us load up! What are we waiting
for?”
It was evident he was wrought far beyond any susceptibility to
moral warning or rebuke. The rector perceived this, I think, and
submitted himself to circumstance.
The truck was hurried up, and the chest placed upon it. It needed
our united efforts to raise the thing; and at our every stagger Joshua
sawed out a little jubilant laugh. We gathered the tools and the ropes
and the ruin of the wrench, and piled all on top. Then we disposed
the broken skeleton amidst, and started on our way home.
It was a hard pull now, though we all gave a hand to it. Three
o’clock had struck, when at last, exhausted and agitated, we drew
the little cart cautiously up to the study window, and unloaded it of its
weightest burden, leaving the rest temporarily outside while we
examined our haul.
The box had been stoutly fastened and secured; but the wood
being shrunk away from its clamps rendered our task an easy one. A
little wrenching with forceps, and the whole lid came apart, sinking
upon the floor with a dusty clang. And then——
 Sleeking and glinting through a dust of perished rags—piled to the
throat, and kept burnished by the sand that had filtered in—a glut of
gold!
Gold in rouleaux and ingots; gold in sovereigns and ryals; gold in
angels and rose-nobles—near all of Henry the Seventh’s and Henry
the Eighth’s reigns, and of incalculable antiquarian, apart from their
intrinsic, value; gold in patens; gold and more in a jewelled ciborium;
chased gold and ivory in an exquisite chalice with handles, and little
queer figures of saints in rich enamel; gold in such wealth as we had
never dreamt.
The vessels had been wrapped, it appeared, in soft skins of
suckling-calf vellum, which had long crumpled into a floury meal,
keeping all bright as blossoms preserved in sand, and easy to dust
and blow away, We felt fairly drunk with the sight, as we gazed down
spell-bound into that brimming reservoir of all wealth.
And then suddenly Mr. Sant had fallen upon his knees.
“O Lord!” he prayed, in a low half-agonized tone; “teach thy
servant to deal rightly with this, converting it to fair uses, and
justifying himself of Thy generosity.”
A little dead silence followed; and at the end Joshua bowed his
head, and raising his hands clasped together, cried twice, in a firm
voice—
“Amen!”
And so at last was consummated that wonderful and tragic tale of
mystery and fatality, which had begun for me in the old court house
of Ipswich. Truly, other things than hanging and wiving go by destiny.
 CONCLUSION.
There was a sequel, which I must relate. Stories of recovered
treasure, if true like this, do not always end with the emotional unities
and the final chapter. Morning does not always bring a confirmation
of pious resolves. A little sourness of digestion sometimes impairs
the glamour of last night’s feast of righteousness. That is the deuce
of it.
Now, I will not say that Joshua repudiated in the slightest reality
the sense of that “Amen” of his; but, once awake and restored to the
full realization of his possession, he certainly did try to back out of
his undertaking to challenge the law to deprive him of it. Not
unscrupulously—not in the least. He merely strove to convince Mr.
Sant as to the actual letter of that law, and, consequently, of the
Quixotry of calling upon it to establish his claim—probably at
considerable expense to both sides—to do what was already, by its
own decreeing, indubitably his.
But he was entirely unsuccessful. The rector, seeing in this only a
personal obstructive policy, designed to shackle that main moral
question of the cleansing of his Augean stable, utterly declined to
forego his bond, and wrung a promise out of my reluctant relative
himself that I should not be allowed to touch a penny of this treasure
until it could be proved well-gotten.
So Joshua, forced at last to give way, though with a very ill grace,
sent in his notice to the Ipswich coroner.
In the mean time the process of cleansing was carried through
with all despatch. The hill was cleared, at some risk, of its tragic
impedimenta, which—after a jury had sat on them, and brought in a
verdict of accidental death—were consigned to rest in the
churchyard—Abel’s, with some distinction, in a separate grave. The
whole story was wrung out at the inquest, and aired, and hung up on
the lines for gossips to find holes in; and gradually the village—with
the entire country-side, to boot—subsided from its fever heat of
excitement, which was only to suffer a temporary recrudescence in
the cause célebre which came presently to provide the epilogue.
One day, a tax-cart, a coroner’s clerk, a posse of insurance-office
firemen, and a couple of cavalrymen from the barracks to escort the
whole, appeared before the rectory, and, removing the treasure-box,
well encased and sealed, from the clerical strong-room—where it
had lain perdu since its discovery—mounted that and Joshua in the
vehicle, and incontinently drove away with both.
We saw him go, sitting darkly on the top of his coffin, like a
dyspeptic Jack Sheppard being jogged off to Tyburn; and thereafter
for a desperate week or more heard or saw nothing of him. Then one
day, a great trumpeting and cheering in the street brought us all out
pell-mell; and there he was, worshipful in the repute of fabulous
riches, being carried shoulder high.
He had won his cause; and through whom do you think? Why, Mr.
Quayle. The little Q.C. accompanied the procession, and shared in
its triumph. Joshua had alighted on him, quite accidentally, in
Ipswich, and revealing to him everything—not without an ironic
satisfaction, one may be sure, in returning at this eleventh hour a
Rowland for his Oliver—had engaged him to conduct his case. And
he had done it, and won it; and the treasure was ours.
“Out of the mouths of babes and sucklings,” said the little man,
meeting me again with delight. “Richard, I am rebuked. I once said
you were the son of your father, but not so good a lawyer. I withdraw
the riservation, entirely. You could see further than some of us into a
stone wall. To think now that your friend spoke the truth through ut
all! I’ll never trust the evidence of me nine senses again. Five, is ut?
Well, I was thinking of the Muses, I suppose. ’Tis a weakness I have,
and will prove my undoing in the end. Never you bother about the
girls, Richard. They spoil your law.”

I have only a word or two to add. I am afraid to declare what that

box of gold realized. The sum, anyhow, was so large as to enrich us
all. A great part of its treasures was distributed into the cabinets of
collectors, the beautiful chalice finding its way, I believe, at an
immense figure, into the museum of a famous cardinal and virtuoso
in Rome. From the total proceeds Joshua handsomely presented to
Harry the equivalent of a comfortable income, which was the means
of helping my dear friend to the very satisfactory position to which he
attained a few years later in London. For me he held the residue
nominally in trust till I was come of age, when he proposed to
establish himself and Uncle Jenico as pensioners on my bounty. The
question was one merely of terms. We made, in fact, our common
home together until the end, even after I had so far neglected Mr.
Quayle’s advice as to bother my head very much indeed about one
girl, and to wive her into the bargain.
We had left Dunberry soon after the events narrated above, taking
Mrs. Puddephatt with us for housekeeper, and not forgetting Fancy-
Maria. For some time, I understand, after our departure, the famous
crypts were a gazing-stock, attracting so many visitors that in the
end Mr. Sant’s dearest wish was realized, and a popular watering-
place established on the foundations of the old smugglers’ haunt.
But long before that the vaults had been closed, as unsafe, by
councillors’ authority; and at this day only a deep depression in the
soil above denotes the spot under which the tragedy of Abel Pilbrow
was enacted.
So the old order changes—all, that is to say, but Uncle Jenico,
who is engaged at this moment, very bent and white, in
demonstrating to my little boy the method of his latest machine for
solving the riddle of perpetual motion.

THE END
 TRANSCRIBER’S NOTES.
Minor spelling inconsistencies (e.g. drowzily/drowsily,
schoolhouse/school-house, barn-door/barn door, etc.) have been
preserved.
Alterations to the text:
Assorted punctuation corrections.
[Part I/Chapter I]
Change “mamma said we must restrench, and cried” to retrench.
[Part II/Chapter VIII]
“watch him fattening, and enjy him in anticipation” to enjoy.
[Part II/Chapter X]
“He stood before me, dropping wet, a most wretched” to dripping.
[Part II/Chapter XV]
“It was a brilliant moonlight night” to moonlit.

[End of Text]
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