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Introduction Methods
Introduction Methods
I. Introduction
II. Methods
I. Tissue
preparation
II. Observation
III. Histochemistry
Histology
The study of the organization of cells and
extra-cellular material into tissues and
organs.
Tissues
A group of similar cells that usually has a
common embryonic origin and is
specialized for a particular function.
Types of Tissues
Epithelial Tissue
Covers body surfaces, lines cavities; glands
Connective Tissue
Supporting tissue
Nervous Tissue
Excitability
Conductivity
Muscular Tissue
Contractility
Biopsy
Removal of a sample of living tissue for
microscopic examination.
Used to diagnose cancers, infections, etc
How to generate histology slides?
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.
Dehydration
to remove fixative and water from the tissue and replace
them with dehydrating fluid.
There are a variety of compounds many of which are
alcohols. several are hydrophilic so attract water from
tissue.
To minimize tissue distortion from diffusion currents,
delicate specimens are dehydrated in a graded ethanol
series from water through 10%-20%-50%-95%-100%
ethanol.
In the paraffin wax method, following any necessary post
fixation treatment, dehydration from aqueous fixatives is
usually initiated in 60%-70% ethanol, progressing
through 90%-95% ethanol, then two or three changes of
absolute ethanol before proceeding to the clearing stage.
Types of dehydrating agents:
Ethanol, Methanol, Acetone.
2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
4 Using warm forceps select the tissue, taking care that it does not cool in
the air; at the same time.
5- Chill the mould on the cold plate, orienting the tissue and firming it into
the wax with warmed forceps. This ensures that the correct orientation is
maintained and the tissue surface to be sectioned is kept flat.
7- Cool the block on the cold plate, or carefully submerge it under water
when a thin skin has formed over the wax surface.
multiple tissue pieces are aligned across the long axis of the mould,
and not placed at random
Processing methods and routine
schedules
Machine processing
Manual processing
CUTTING
using the microtome
A microtome is a mechanical instrument
used to cut biological specimens into very
thin segments for microscopic
examination. Most microtomes use a steel
blade and are used to prepare sections of
animal or plant tissues for histology. The
most common applications of microtomes
are
Microtome knives
STEEL KNIVES
NON-CORROSIVE KNIVES FOR
CRYOSTATS
DISPOSABLE BLADES
GLASS KNIVES
DIAMOND KNIVES
1- Traditional histological technique:
tissues are hardened by replacing water with paraffin. The tissue is
then cut in the microtome at thicknesses varying from 2 to 25
micrometers thick. From there the tissue can be mounted on a
microscope slide, stained and examined using a light microscope
2- Cryosection:
water-rich tissues are hardened by freezing and cut frozen;
sections are stained and examined with a light microscope.
This technique is much faster than traditional histology (5
minutes vs. 16 hours) and are used in operations to achieve a
quick diagnosis. Cryosections can also be used in
immunohistochemistry as freezing tissue does not alter or
mask its chemical composition as much as preserving it with a
fixative.
STAIN and MAINTAIN Structural Integrity
Hematoxylin and Eosin (H & E)
H & E is a charge-based, general purpose stain. Hematoxylin
stains acidic molecules shades of blue. Eosin stains basic
materials shades of red, pink and orange. H & E stains are
universally used for routine histological examination of tissue
sections.
Fixation
Any well fixed tissue.
Staining Procedure
1- Deparaffinize and hydrate to water
2- If sections are Zenker-fixed, remove the mercuric chloride crystals
with iodine and clear with sodium thiosulphate (hypo)
3- Mayer's hematoxylin for 15 minutes
4- Wash in running tap water for 20 minutes
5- Counterstain with eosin from 15 seconds to 2 minutes depending
on the age of the eosin, and the depth of the counterstain desired.
For even staining results dip slides several times before allowing
them to set in the eosin for the desired time
6- Dehydrate in 95% and absolute alcohols, two changes of 2
minutes each or until excess eosin is removed. Check under
microscope
7- Clear in xylene, two changes of 2 minutes each
8- Mount in Permount or Histoclad
Results
Nuclei - blue - with some metachromasia
Cytoplasm - various shades of pink-identifying different tissue
components
Renal nephron
http://www.meridianinstitute.com/eamt/files/burns2/54burns2.jpg
nephrotic range proteinuria-proximal tubule
Pathology http://www.gamewood.net/rnet/renalpath/ch1.htm
Special situations
Staining – routine stain – H&E
Some structures are seen/ preserved (large molecules like
nucleoproteins, cytoskeleton proteins, ECM proteins- collagen,
membrane proteins)
some are not seen/lost (small molecules -t-RNA, large molecules like
glycogen & Proteioglycans are dissolved, )during the fixation/staining
process
Special fixatives to retain membrane ( phospholipids)
Permanganate & osmium – for EM
For Elastic fibers – Orcein/ Resorcin – Fuscin
For reticular fibers – Silver impregnation
Histochemistry & Cytochemistry
Specific binding of dye with particular molecule
Fluorescent dye labeled antibody to cell component
Enzyme activity
Autoradiography – radio isotopes tagged with precursors of a
molecule molecule incorporated into cell/ tissue before fixation
H&E, Hematoxylin and Eosin
•Hematoxylin stains basophilic
structures
•Eosin stains acidophilic
structures
Hematoxylin- nuclei; eosin- cytoplasm
Gomori trichrome stain
http://freepages.genealogy.rootsweb.ancestry.com/~gomery/gomorigeo.html
www-bioc.rice.edu/bios576/immuno/Trichrome.jpg
Special stain
PAS positive substances Carbohydrate
(glycogen) or carbohydrate rich molecules,
Basement membrane, reticular fibers
Periodic acid cleaves bond between carbon atoms
form aldehyde group
Aldehyde binds with Schiff to produce magenta or
pink color
Periodic Acid Schiff’s stain (PAS)- glycogen, mucopolysaccharides
Feulgen stain for Nuclear Proteins
Acid hydrolyses or cleaves proteins from
deoxyribose of DNA leads to opening of
sugar group & formation of aldehyde
Schiff binds and gives magenta color to
aldehyde
Can be useful to quantify amount of DNA ( by
using spectrophotmetry of Feulgen stained
tissue)
3
Figure 1—16. Photomicrograph of
a bone section treated with a
histochemical technique to
demonstrate calcium ions. The
dark precipitate indicates the
presence of calcium phosphate in
calcified bone and cartilage.
Noncalcified cartilage tissue
(stained in pink) is in the upper
portion of the figure. Medium
magnification.
Immuno Histo Chemistry (IHC)
Antibody ( Immunoglobulin) conjugated with
fluorescent dye( most common is Fluorescein)
+ Antigen ( foreign protein)
Fluorescein absorbs UV light and emits
green fluorescence can be seen under
Fluorescent microscope (IF- Immuno
Fluorescence)
Example :- actin (Antigen) of Rat infected
to Rabbit blood of Rabbit ( have poly -
clonal antibodies for Rat’s actin/ anti rat actin
antibodies) bind with Fluorescent dye
Monoclonal Antibodies
Specific antigen Multiple Myeloma pts.
(actin of rat)
↓
B lymphocytes of
Immunized rabbit
Monoclonal B ells
Hybridoma cells
↓
Single specific type of antibodies (Monoclonal)
( against Actin)
Clinical Significance of Monoclonal
Antibodies
Diagnosis of tumors(tumor markers) &
Infections( HIV, Infectious Mononucleosis)
Classify sub – types (B -cell and T- cell
lymphomas)
Treatment – Anti-TNF-α antibodies in
inflammatory disorders
Direct Immunocytochemistry
Indirect Immunocytochemistry
2nd antibody employed
Figure 1—26. Photomicrograph
of a section of small intestine in
which an antibody against the
enzyme lysozyme was applied
to demonstrate lysosomes in
macrophages and Paneth cells.
The brown color results from
the reaction done to show
peroxidase, which was linked to
the secondary antibody. Nuclei
counterstained with
hematoxylin. Medium
magnification.
Figure 1—28. Electron micrograph showing a section of a pancreatic acinar cell
that was incubated with anti-amylase antibody and stained by protein A coupled
with gold particles. Protein A has high affinity toward antibody molecules. The
gold particles appear as very small black dots over the mature secretory granules
and the forming granules in the Golgi complex. (Courtesy of M Bendayan.)
Enzyme Histochemistry
Localization of enzymatic activity in tissues
Best fixation – mild aldehyde ( formalin)
Basis – localized reaction production of
enzyme activity
Used for acid & alkalineenzyme
phosphatase, ATP
ases
AB (substrate) + T (trap) AT (
reaction product) + B (Hydrolyzed
component of substrate)
Other Methods