STAINING

• The process of applying dyes on the sections to see METHODS OF STAINING

and study the architectural pattern of the tissue and DIRECT STAINING:
the physical characteristics of the cells under the • A process of giving color to
microscope. the sections by using
✓ Tissues and cells become more visible aqueous or alcoholic dye
✓ Morphologic changes are more easily identified solutions.
✓ Presence or absence of disease processes can INDIRECT STAINING:
be established. • A process whereby the
Nucleus action of the dye is
• acidic in character intensified by adding
• have greater affinity for basic dyes another agent or mordant.
• basophillic
Cytoplasm MORDANT
• basic constituent of the cell • Serves as a link or bridge between the tissue and
• have greater affinity and take more of the the dye, to make the staining reaction possible.
acid stains • It combines with a dye to form a colored "lake",
• acidophillic which in turn combines with the tissue to form a
"tissue-mordant-dye-complex" that is rendered
In general, microscopic examination is facilitated if two insoluble in ordinary aqueous and alcoholic
contrasting stains are used solvents.
• It is an integral part of the staining reaction itself,
HEMATOXYLIN without which no staining could possibly occur.
- stains the nuclear detail of
the cell ACCENTUATOR
EOSIN • Accelerates or hastens the speed of the staining
- brings out the cytoplasmic reaction by increasing the staining power and
detail of the cell and the selectivity of the dye.
tissue's architecture.

For the purpose of simplicity, staining of tissues can be

classified into three major groups, namely:
a. HISTOLOGICAL STAINING
Tissues constituents are demonstrated in sections PROGRESSIVE STAINING:
by direct interaction with a dye or • A process whereby tissue
staining solution, producing coloration elements are stained in a
of the active tissue component. H & E Staining definite sequence and the
staining solution is applied
b. HISTOCHEMICAL STAINING (HISTOCHEMISTRY) for specific periods of time
Various constituents of tissues are or until the desired intensity of coloring of the
studied through chemical reactions different tissue elements is attained.
that will permit microscopic • Once the dye is taken up by the tissue, it is not
localization of specific tissue washed or decolorized.
Congo Red Staining
substances. • The differentiation or distinction of tissue detail
relies solely on the selective affinity of the dye for
c. IMMUNOHISTOCHEMICAL STAINING different cellular elements.
Combination of immunologic and histochemical
techniques that allow phenotypic markers to be REGRESSIVE STAINING
detected and demonstrated under the microscope, • With this technique, the
using a wide range of polyclonal or tissue is first overstained to
monoclonal, fluorescent labeled or obliterate the cellular
enzyme-labeled antibodies to details, and the excess stain is removed or
detect and demonstrate tissue HCG Staining decolorized from unwanted parts of the tissue, until
antigens. the desired intensity of color is obtained.
DIFFERENTIATION (DECOLORIZATION) COUNTERSTAINING
• Selective removal of excess stain from the tissue • Counterstaining is the
during regressive staining in order that a specific application of a different
substance may be stained distinctly from the color or stain to provide
surrounding tissues. contrast and background
Counter staining
to the staining of the
Differential Staining structural components to be demonstrated.
-Uses more than one chemical
stain to better differentiate METALLIC IMPREGNATION
between various microorganisms Gram Staining • Metallic Impregnation is a
or structures/cellular components process where specific tissue
of a single organism. elements are demonstrated,
-This is usually done by washing the section in a simple not by stains, but by colorless
solution (e.g. water or alcohol), or by the use of acids solution of metallic salts Metallic Impregnation:
brain parenchyma
and oxidizing agents. which are thereby reduced by
the tissue, producing an opaque, usually black
METACHROMATIC STAINING deposit on the surface of the tissue or bacteria.
• Metachromatic staining technique entails the use of
specific dyes which differentiate particular VITAL STAINING
substances. • Vital staining is the selective staining of living cell
• Water is necessary for most metachromatic staining constituents, demonstrating cytoplasmic structures
techniques, and metachromasia is usually lost if by phagocytosis of the dye particle (cytoplasmic
the section is dehydrated in alcohol after staining. phagocytosis).
• Although it is preferable to use frozen sections of • The nucleus of the living cell is resistant to vital
fresh or rapidly fixed tissues, satisfactory stains, and therefore is not demonstrated.
metachromasia may also be attained in formalin- • If nucleus is demonstrated during vital staining
fixed tissues suggests permeability of the membrane of the dye,
signifying the death of the cell.
METACHROMASIA
－ The property to stain biological materials with a INTRAVITAL STAINING
color that is different from that of the stain itself. • Intravital staining of living cells is done by
injecting the dye into any part of the animal
body (either intravenous, intraperitoneal or
subcutaneous), producing specific coloration of
certain cells, particularly those of the reticulo-
Albert’s stain (green) -
endothelial system.
Toluidine blue – mast cells
(green) Corneybacterium diphtheriae
tail end blue

Metachromatic dyes are basic dyes belonging to the

thizine and triphenylmethane groups, such as:
1. Methyl violet or crystal violet
2. Cresyl blue
3. Safranin • Common dyes used are:
4. Bismarck brown ✓ Lithium
5. Basic fuchsin ✓ Carmine
6. Methylene blue ✓ India ink
7. Thionine
8. Toluidine blue SUPRAVITAL STAINING
9. Azure A, B, C • Used to stain living cells
immediately after removal
from the living body. Brilliant Cresyl
• Thin slices of tissues are placed Blue: Reticulocytes

in small staining dishes and enough staining

solution is added to cover the tissue.
 Common dyes used are:
✓ Neutral red
✓ Janus green
✓ Trypan blue
✓ Thionine
Janus green
Paraffin wax is poorly permeable to most
staining solutions and should therefore be
removed from the section prior to staining
“Sections of Water”
• The alcohol is then finally replaced with water
before actual staining of section is performed.
“Sections to Alcohol”
After deparaffinization with xylene, the section is
subjected to decreasing grades of alcohol, and in such
instances, the term is used.
*It is not advisable to let the section stay in alcohol for a
long time because many stains are usually removed by
prolonged immersion in alcohol.
For staining, several materials are
needed, including:
1. COPLIN JAR
-slotted jar holding from 5 to 9 slides
2. SLOTTED STAINING DISHES
• holding from 5 to 19 slides
over which different
solutions are poured.
• Slides are placed on end
singly or in staggered fashion, in the arm.
3. METAL OR GLASS STAINING RACKS OR CARRIERS
• holding from 10-30 slides upright.
• Slides are transferred to appropriately sized glass
dishes containing the staining solutions
Routine Hematoxylin and Eosin (H&E) staining
-is the most common method utilized for
microanatomical studies of tissues, using the regressive
staining which consists of overstaining the nuclei,
removal of superfluous and excessive color of the
tissue constituent by acid differentiation
-H&E Staining is the cornerstone of tissue-
based diagnosis
ROUTINE H&E STAINING in Paraffin Section
(Regressive Staining) Procedure:
FIXATION
• Most fixative can be used except osmic acid
solutions which inhibit hematoxylin.
PROCEDURE:
1. Clear paraffin embedded sections in the first
xylene bath for 3 minutes.
2. Transfer to a second xylene bath for 2 to 3
minutes.
3. Immerse in the first bath of absolute ethyl
alcohol for 2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 or
2 minutes.
5. Rinse in running water for 1 minute
*removal of pigments is done after rehydration
and right before staining.
6. STAIN with Harris Alum hematoxylin for 5
minutes (Ehrlich's hematoxylin requires 15-30
minutes)
7. Wash in running tap water to remove excess
stain.
8. Differentiate in 1% acid alcohol (1 ml.
concentrated HCl to 99 ml. of 80% ethyl alcohol)
for 10-30 sec. controlling microscopically until
only the nuclei are stained.
9. Rinse in tap water.
 10. Blue in ammonia water (average of 5 minutes)
or 1% aqueous lithium carbonate until the
section appear blue (about 30 seconds)
11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5
minutes. If alcoholic eosin is used, the tie can
be reduced to 30 seconds or 1 minute.
13. If aqueous eosin is used, wash and
differentiate in tap water under microscope
control until the nuclei appear sharp blue to
blue black and the rest of the tissue in shades of
pink. If an alcoholic solution is used,
differentiate with 70% alcohol.
14. Dehydrate, clear and mount.
Results: Cell nuclei >> blue
Other constituents colored according
to the counterstain used.
3. Malory's Phloxine Methylene Blue
Stain
• Originally known as Eosin Methylene Blue(EMB)
method
• This technique produces a sharp nuclear stain and
reveals with marked differentiation the various
structures in the tissues, which should be fixed in
Zenker's fluid.

Mallory's Phloxine B-Methylene Blue-Azure II Stain Emphasizes

Elastin and Collagen Bundles in Epoxy Embedded Lung

H&E staining of Frozen Sections for Rapid Diagnosis

(Progressive Staining)
Four staining methods are commonly employed for
Nuclei Blue to blue black
frozen sections, the choice depending upon the
personal preference of the pathologist and the type of
Karyosome Dark blue tissue section to be stained.
1. Hematoxylin-Eosin method
Cytoplasm, proteins in edema fluid Pale pink
2. Thionine method
RBCs, eosinophilic granules, keratin Bright orange-red 3. Polychrome Methylene Blue method
Basophil cytoplasm, plasma cells & 4. Alcoholic Pinacyanol method (used also for
Purplish pink
osteoblast supravital staining of Mitochondria and
Pink or light blue to dark primarily for color sensitization in photography
Cartilage
blue
PROCEDURE:
Calcium and calcified bone Purplish blue
1. Orient section in the block and freeze with
Decalcified bone matrix, collagen & liquid nitrogen.
Pink
osteoid
2. Cut cryostat sections at 5-10 micra.
Muscle fibers Deep pink 3. Mount sections on to albuminized slides and
dip in a 10% formalin to fix.
H&E Staining Technique 4. Rinse rapidly in water.
1. Heidenhain's Iron Hematoxylin Method 5. Stain with Harris hematoxylin for 30-45
Results: seconds.
Cells nuclei, cytoplasmic 6. Rinse in tap water.
inclusions and muscle 7. Blue in ammonia water for 5 seconds. Rinse in
striations >> black tap water.
Other constituents are 8. Counterstain with 5% aqueous eosin or 1%
colored according to alcohol eosin for 1 minute.
counterstain. 9. Rinse in tap water.
10. Dehydrate in increasing concentration of
2. Celestine Blue-Haemalum Sequence Staining alcohol.
• Celestine blue is an oxazine dye used as an 11. Clear with xylene.
alternative to iron hematoxylin nuclear stain, 12. Mount with cover slide
producing a strong and precise nuclear stain that is
resistant to decolorization by succeeding acid stains
and solutions.

Rapid Metachromatic Staining of Frozen Sections
(Eye Dropper Method)
• Frozen sections mounted on the slides may be
stained as in paraffin sections although the duration
of staining is usually shorter.
• Frozen sections may be stained by picking up
sections on albuminized slides and drying them
quickly or by simple direct staining on a wet slide
with an eye dropper.
Meaning we only use a single stain: Metachromatic
stain only
Collodionization
• Coating the slide with
dilute (thin) celloidin
solutions permits firm
attachment of paraffin
ribbons.
• This is also recommended for sections that will be
subjected to strong alkaline or acid solutions and
for tissues that contain glycogen for demonstration.
• Cellulose nitrate (celloidin) is soluble in absolute
alcohol, hence, treatment with absolute alcohol
alone should be avoided during dehydration and
clearing of stained sections
• The celloidin will be removed in the final
dehydration with absolute alcohol prior to clearing
and mounting.
Precautions on Staining
1. Stains on the skin should be avoided not only
because they are signs of poor technique but because
stains are health hazards per se, being slowly absorbed
by the skin and eventually producing side effects.
• Stains may be effectively removed from the skin by
prompt topical application of 0.5% acid alcohol,
followed by rinsing with tap water.
2. Failure of the section may be due to paraffin, fixative,
or decalcifying solution that has not been thoroughly
washed out or removed.
• Alternatively, if the section does not stain, the
staining solution may be faulty. Hematoxylin
solution may not have been properly and
sufficiently ripened. (Hematoxylin must not be used
too soon after preparation to ensure complete
ripening.
3. If after staining, sections are fuzzy and do not appear
clear under the microscope, xylol should be
replenished.
• To remedy the condition, the section is placed
in a Coplin jar containing xylol to dissolve the
adhesive. The slide is run back through the
various processes up to the point where the
fault was; a fresh solution is used, and the
tissue is re-stained.
4. Stains may be saved and used again for as long as
they have not lost their staining properties. Sections
are usually rinsed with distilled water before placing
them in used stains.
• Formation of precipitate in staining solution
and poor staining results signify loss of staining
property and hence, the stain should be
discarded and replaced with a fresh solution.
5. Failure of sections to remain on the slide during
staining could have been due to dirty or oily slide.
• Albumin fixative may be too old, as suggested
by the loss of its clear color or emission of an
odor. To avoid this, adhesives should be
prepared in not too great amounts (around 1
ounce) which may last for 2-3 months.
Re-staining of Old Sections
Old, bleached or faded sections may be restained:
1. The slide is usually immersed in xylene for 24
hours, or gently heated until the mounting
medium begins to bubble.
2. The coverslip may then be removed by lifting it
with a dissecting needle.
3. The section is placed in xylene for 30 minutes
to remove the remaining balsam and then
brought down to water.
4. It is placed in a 0.5 potassium permanganate
solution for 5-10 minutes, rinsed in tap water
and subsequently immersed in 5% oxalic acid
for 5 minutes or until the section is decolorized.
5. After washing it again in running tap water for
another 5 minutes, the section may then be re-
stained with the appropriate staining
technique.
Broken Slides Stains and Staining Solutions
❖ Mounting a broken Biological stains or coloring substances are prepared
slide on to another from dyes which may generally be divided into two
clean xylene-moist categories:
slide with a drop of
mounting media A. NATURAL DYES
(Clarite or Permount) Natural dyes are those obtained from plants and
may be sufficient for animals, previously utilized for dyeing of wool and
immediate cotton. Among the most common natural dyes available
examination while a are:
new section is being 1. HEMATOXYLIN -heartwood of Mexican tree
cut and stained. known as “Hematoxylin Campechianum”.
❖ If an important slide 2. COCHINEAL DYES - is the most important of
is broken and the insect dyes and comes from the bodies of
replacement is not female insects
available, the section 3. ORCEIN
(is still intact) may be 4. Saffron
transferred to
another slide. B. SYNTHETIC (ARTIFICIAL) DYES
known as "Coal Tar Dyes
They are derived from the hydrocarbon benzene (C6H6),
and are collectively known as Aniline Dyes.

Natural Dyes
1. HEMATOXYLIN
• powerful nuclear and
chromatin staining capacity,
and its striking polychrome
properties
• not a true basic dye
• HEMATEIN - the active coloring agent
-formed by the oxidation of hematoxylin, a
process known as "ripening".

RIPENING
➢ Natural Ripening
• exposing the substance to air and sunlight
➔oxidizing hematoxylin.
• Such a process is slow and takes as long as 3-4
months
➢ Artificial Ripening
• Ripening can be accelerated by adding strong
oxidizing agents such as:
2. COCHINEAL DYES Synthetic Dyes
• An old histologic dye extracted A. Chromophore
from the female cochineal bug, • Are substances with definite atomic groupings
which is treated with alum to and are capable of producing visible colors.
produce the dye, carmine. B. Chromogens
• It is widely used as a powerful • Simple benzene compounds which contain
chromatin and nuclear stain for fresh material and chromophores.
smear preparations. • the color they impart to the tissue is not
Picrocarmine permanent
when combined with picric acid, it is extensively • Before a chromogen can properly be called a
used in neuropathological studies; dye, it must have the property of retaining its
color in the tissue. This property is acquired by
Best’s Carmine Stain the addition of an auxochrome.
when combined with aluminum chloride, is used C. Auxochrome
for the demonstration of glycogen. An auxiliary radical or substance which imparts to the
compound the property of electrolytic dissociation,
3. ORCEIN thereby altering the shade of the dye, enabling it to
• A vegetable dye extracted form salts with another compound, and ultimately
from certain lichens which retaining its color.
are normally colorless.
• When treated with: A dye therefore, should consist of a chromophore and
✓ ammonia an auxochrome group attached to a hydrocarbon
✓ exposed to air benzene ring.
*produces blue or violet
colors. Orcein: Elastic fiber a DYE molecule
It is a weak acid, is soluble in alkali, and is mainly used
for staining elastic fibers

Litmus is also obtained from

lichens, treated with lime and
soda, and exposed to ammonia
and air.
-not used as a cytological stain because of its
poor staining property.
-used mainly as an indicator. The coloring property is attributed to the chromophore.
The dyeing property is attributed to the salt-forming
4. Saffron auxochrome.
Saffron is a spice derived from
the flower of Crocus sativus, Depending on where the coloring substance
commonly known as the (chromophore) is found, dyes may be classified into
"saffron crocus". three groups:
Saffranin: An orange-red ❖ ACID DYES
dyestuff extracted from the saffron. ❖ BASIC DYES
❖ NEUTRAL DYES

ACID Dyes
• The active coloring substance is found in the acid
component, and the inactive base, e.g. acid fuchsin,
is usually sodium salt of a sulfonate of rosaniline.
• Basic cell structures have an affinity for the acid dye
ions and are regarded as acidophilic.
One example of such dye is Picric acid, which has the • Ethyl alcohol or acetic acid-fixed tissues, on the
ability to form salt with an other hand, readily take in both basic and acidic
alkali. dyes.
- outstanding in the sense Examples:
that it is the only substance so Romanowsky dyes used in hematology,
far that can fix, differentiate or Giemsa's stain
stain tissue all by itself. Irishman's stain for leukocyte differentiation
- It may also be used as a fixative, as a decalcifying
agent or as a tissue softener.
- counterstain to basic cytoplasmic stains, to acid
Romanowsky dyes:
fuchsin in Van Gieson's connective tissue staining, or to Bronchoalveolar lavage

crystal violet for the study of fungi Giemsa's stain

Common Staining Solutions used

A. HEMATOXYLIN
• for routine histologic studies.
• The mordant : alum and iron, forming lakes or
colored complexes (dye-mordant-tissue complexes).
The color of which will depend on the salt used.
• Aluminum Salt Lake - are usually colored blue
BASIC Dyes
• Ferric Salt Lakes - are colored blue-back
• The active coloring substance is found in a basic
component that combines with the acid radical
(usually taken from sulfuric, acetic or hydrochloric
acid).
• Acidic cell structures have an affinity for basic dye
ions and are therefore regarded as basophilic.

Methylene Blue
• a basic nuclear stain
• may be used both as an
indicator or a dye
• very widely used in
microbiology for bacterial Aluminum Hematoxylin SOLUTIONS
staining • Recommended for progressive staining of
The Phosphate Backbone of tissues, (i.e. staining for predetermined time to
the DNA is responsible for adequately stain the nuclei but leave the
its ACIDIC Nature
background tissue relatively unstained, to be
later counterstained with eosin, congo red or
safranin).
• The alum hematoxylin can also be used for
regressive staining, meaning that the section is
Methylene Blue: bacteria
overstained, and then differentiated in acid
alcohol followed by "blueing".
NEUTRAL Dyes The two main alum hematoxylin solutions employed
• formed by combining aqueous solutions of acid and are:
basic dyes, capable of staining cytoplasm and • Ehrlich's hematoxylin- ripening is brought by
nucleus simultaneously and differentially. the addition of sodium iodate
• Because they are made up of large molecular • Harris hematoxylin solutions - ripened with
complexes, neutral dyes are insoluble or barely mercuric oxide
soluble in water, but they are usually soluble in
alcohol.
BLUEING
• done after differentiation with acid alcohol
In the presence of excess acid, aluminum hematoxylin
dye-lake fails to form due to lack of -OH ions = RED
• During staining, alum hematoxylin stained
sections are usually passed on to an alkaline
solution (e.g. 1% hydroxide) in order to
neutralize the acid and free the -OH group, to
form an insoluble blue aluminum hematin-
tissue lake. Such procedure is known as
BLUEING.
1.Ehrlich's Hematoxylin – Steps:
1. Dissolve hematoxylin in absolute ethyl alcohol
with gentle heat.
2. Dissolve the potassium alum in distilled water
and glycerin with gentle heating and shake.
3. Mix the two solutions and add glacial acetic
acid.
4. Ripening: Exposed to air and sunlight for
several weeks or months in a flask lightly
plugged with cotton shaking daily.
5. Transfer in a well-stoppered bottle and store in
a warm place.
naturally ripening alum hematoxylin takes about 2 months to ripen,
but its staining property will last for months or years.
Glycerin
• added to slow the oxidation process and
prolong the shelf life of hematoxylin
• acts as a stabilizer, retards evaporation of the
solution, and appears to slow down ripening,
so that it may be added 4-6 weeks after the
initial preparation.
2. Harris Hematoxylin
• a good regressive stain that may either be used
immediately or stored for future use, since it
remains stable for a long time (about 6
months).
• Widely used for:
✓ routine nuclear staining,
✓ exfoliative cytology
✓ staining of sex chromosomes. Barr body
• The usual staining time is 5-20 minutes,
depending on the batch and age of stain, the
nature of tissue, and the degree of staining
required.
• Ripened by HgO, solution will assume a dark
purple color
• Preservative - Ethyl alcohol
• precipitates form on prolonged storage, must
be filtered off before use
3. Cole's Hematoxylin
• recommended for routine purposes, especially
used in sequence with celestine blue.
• It is ready for immediate use, but may need
filtering after storage, as with Harris
hematoxylin.
• Ripened by: alcoholic iodine solution
4. Mayer's Hematoxylin
• an alum hematoxylin which can be used as a
regressive and progressive stain
• nuclear counterstain to demo the presence of
cytoplasmic glycogen by special stain.
• Ripened by: sodium iodate
IRON HEMATOXYLIN
• Iron hematoxylin compounds are used only for
differential or regressive staining, using acid alcohol
as a differentiating agent.
Two main iron hematoxylin solutions are employed for
routine work in the laboratory:
1. Weigert's solution: using ferric ammonium chloride
as mordant
• standard iron hematoxylin for demonstrating
muscle fibers and connective tissues.
• recommended when the preceding stains
contain acid (e.g. Van Gieson stain containing
picric acid) which decolorizes nuclei stained
with alum hematoxylin.
Counterstained with Von Gieson
2. Heidenhain's solution: using ferric ammonium
sulfate (iron alum) as mordant
• The dye lake obtained
when ferric salts are used
as mordants is an intense
blue-black one.
Intralobular duct lined by cuboidal cells Heidenhain's iron hematoxylin, red
arrow – basal pole
3. Regaud's Hematoxylin for Mitochondria • Variants include:
• most permanent and ✓ Wright's stain
simplest method to ✓ Jenner's stain
demonstrate mitochondria by ✓ Leishman stain
light microscopy Mitochondria:
✓ Giemsa stain
small dots

4. Phosphotungstic Acid hematoxylin (PTAH)

• Stains nuclei, fibrin, muscle striations, and
myofibrils blue Other Stains Used:
• Staining is progressive 1. Acid Fuchsin-Picric Acid (Van Gieson's Stain)
Result: • a mixture of picric acid and acid
Collagen, bone and cartilage are fuchsin for the demonstration of
stained orange-red or brownish connective tissues.
red to deep brick-red stain
2. Acid Fuchsin(Masson Stain)
phosphotungstic acid–hematoxylin (PTAH) staining of • stains collagen, smooth muscle, or
formalin-fixed-paraffin embedded human skeletal muscle
mitochondria.
• used as a nuclear and cytoplasmic
B. EOSIN stain in Mallory's trichrome
• one of the most valuable stains used for • collagen stain red with acid fuchsin in Van Gieson's
differentially staining connective tissues and stain
cytoplasm.
• routinely used in histopathology as a counterstain 3. ACRIDINE ORANGE
to hematoxylin and before methylene blue. • a basic acridine fluorochrome
• imparts a pink or red color to cytoplasmic material, which permits discrimination
cell membranes, and some extracellular structures between dead and living cells
used a background stain because it gives a pleasing and • green fluorescence for DNA
colorful contrast to nuclear stains, particularly in • red fluorescence for RNA.
chromate and picric acid fixed tissues, and in acid • useful for cell cycle determination
decalcified materials which are strongly stained with
eosin 4. ACRIDINE RED 3B
*It is red general cytoplasmic acid dye available in three • used to demonstrate deposits of
forms: calcium salts and possible sites of
a. Yellowish (Eosin Y) phosphatase activities.
• most commonly used.
• It is readily soluble in water, less in alcohol, 5. ALCIAN BLUE
available in both aqueous and alcoholic • a complex, water soluble
solutions, showing a green yellow fluorescence phthalocyanine dye, similar
especially in alcoholic medium. to chlorophyll which stains
• The aqueous stain is generally used as 1% acid mucopolysaccharides
solution for 15 seconds to 3 minutes, depending by forming salt linkages
Alcian Blue demonstrating
on the tissue, type of fixative and intensity of with them. acid mucopolysaccharides
(blue) of the colon.
color desired. • an excellent stain -
b. Eosin B (Eosin Bluish/Erythrosin B/imperial red) produces a striking blue color, and it is resistant to
- deeper red color various counterstaining procedures.
c. Eosin S (Ethyl Eosin/eosin alcohol soluble) • It is more specific for connective tissue and
epithelial mucin due to its use as an acid solution.

6. ALIZARIN RED S
• forms an orange-red lake
Eosin Y + Alcohol
with calcium at a pH of 4.2
a. Romanowsky Stains
• based on a combination of eosinate (chemically
reduced eosin) and methylene blue
7. ANILINE BLUE 14. CELESTINE BLUE
• a cytoplasmic stain used • an oxazine dye used as an
for counterstaining of alternative to iron hematoxylin
epithelial sections. nuclear stain
• resistant to strong acid dyes
8. AZOCARMINE • forms a strong staining lake with
• nuclei are deep red; iron alum
cytoplasm is a pale red
15. CONGO RED
• best known as an indicator, but
9. BASIC FUCHSIN may be utilized as a stain for axis
• a plasma stain utilized cylinders in embryos.
also for deep staining of • It is used as a 4% aqueous
acid fast organisms, for solution in Krajian's method of
mitochondria, for staining elastic tissue, amyloid
Congo Red: Amyloid
differentiation of The primary stain used in acid-fast staining,
and myelin.
smooth muscles carbolfuchsin, is lipid-soluble and contains
phenol, which helps the stain penetrate the
with the used of cell wall. 16. CRESYL VIOLET
picric acid. • stains the acidic components of
It is a main constituent of: the neuronal cytoplasm (Nissl
Feulgen's and Schiff's reagent bodies) a violet color
• for the detection of aldehyde * Nissl bodies are discrete Cresyl Violet: Nissl body

Van Gieson's solution granular structures in neurons

• for connective tissues, mucin, and elastic tissue that consist of rough endoplasmic reticulum
staining.
17. CRYSTAL VIOLET
10. BENZIDINE • is a nuclear or chromatin stain
used for staining amyloid in
used for staining frozen sections and platelets
hemoglobin. in blood.
Gentian violet is the staining
11. BISMARK BROWN solution formed by the mixture of crystal violet, methyl
used as a contrast stain for Gram's violet and dextrin
technique, in acid fast and
Papanicolau method, and for 18. ETHIDIUM BROMIDE
staining diphtheria organisms. • intercalates and and stains DNA, providing a
fluorescent red-orange stain.
12. CARMINE • does not stain healthy cells - used
used as a chromatin stain for as a marker for apoptosis in cells
fresh materials in smear • may be used in conjunction with
preparations. It is slightly soluble Acridine Orange in viable cell
in water at a neutral reaction, and counting.
usually kept in ammoniacal solution which changes its
properties due to oxidation. 19. GIEMSA STAIN
It is usually combined with aluminum chloride to stain • is used for staining blood to
glycogen (Best Carmine Solution). differentiate leukocytes.

13. CARMALUM (MAYER'S) 20. GOLD SUBLIMATE

SOLUTION • stain used for metallic
• is mordanted dye acting as a impregnation, made
basic dye and staining acidic up of gold chloride and
substances. mercuric chloride.
*Basta metallic impregnation
gani ➔ Black color
21. IODINE 24. MASSON'S TRICHROME
• The oldest of all stains a three-color staining principle
• Originally used for microscopic study of starch Result:
granules. keratin and muscle fibers = red
• It stains amyloid, cellulose, starch, carotenes collagen and bone = blue/green
Masson’s Trichrome stain:
and glycogen. cytoplasm = light red/pink Airways
• It is widely used for removal of mercuric fixative nuclei = black
artefact pigments, and as a reagent to alter
crystal and methyl violet so that they may be 25. METHYL GREEN
retained by certain bacteria and fungi. • stains chromatin green in
• It may also be used in the form of aqueous or the presence of an acid.
alcoholic solutions • It gives false positive
Gram's Iodine (MORDANT in Gm Staining) reactions with certain
• used to identify and differentiate bacteria secretions such as mucin Methyl Green:
Immunohistochemical
▪ gram-positive = deep blue Counter Stain

▪ gram-negative = pink
• used in the Gram Weigert method of staining 26. METHYLENE BLUE
microorganisms and fibrin in tissue sections. • a common basic nuclear
Lugol's Iodine (Lugol's solution) stain employed with eosin
• a brown solution that • stains acidic parts blue and
turns black in the is a good counterstain with Metheylene Blue:

presence of starches Eosin Y Human skin

• can be used as a cell "POLYCHROMING" involves the

stain - makes the cell oxidation of methylene blue, resulting in loss of methyl
nuclei more visible groups and leaving lower homologues of the dye
• is used as a test for (azures) and deaminized oxidation products (thiazoles).
glycogen, amyloid, • The resulting mixture of methylene blue, azures and
and corpora thazoles is known as polychrome methylene blue.
amylacea.
Cysts of E. histolytica/E. dispar in a
Mallory's Phloxine Methylene Blue Stain, originally
concentrated wet mount stained with iodine. Eosin-Methylene Blue (EMB)
22. JANUS GREEN B • produces a sharp nuclear stain
• is used for demonstrating and reveals with marked
mitochondria during differentiation the various
intravital staining. structures in the tissues.

27. METHYLENE VIOLET

• a metachromatic dye formed
whenever methylene blue is
mitochondrial viability observed by staining heated in fixed alkali or alkali
with Janus Green B
carbonate, coloring nuclei Metheylene Violet:
Amyloid
or leukocytes reddish
23. MALACHITE GREEN purple in the presence of methylene blue
• a weakly basic dye used as a
contrast stain for staining 28. NIGHT BLUE
ascaris eggs and • used as a substitute for carbol fuchsin in acid-
erythrocytes, and as a fast staining.
bacterial spore stain
• it is also used both as a 29. NEUTRAL RED
decolorizer and as a counterstain • a basic dye
recommended
for observing Neutral Red:
cell granules and vacuoles of Ovarian Follicles

phagocytic cell
30. NILE RED
• aka Nile blue oxazone
• a lipophilic stain
• accumulates in lipid
globules inside the cells,
staining them red
31. OIL RED O
• used as a stain for neutral lipids
• a rapid and simple stain
• useful in identifying fat emboli in lung tissue or
clot sections of peripheral blood
Oil Red O:
Normal Lipid staining Increased Lipid staining
32. ORCEIN
• an excellent stain for elastic
fibers
• especially recommended in
dermatological studies due
to its ability to demonstrate
the finest and most delicate fibers in the skin
33. OSMIUM TETROXIDE
• used to stain fat,
although other
substances are also
stained simultaneously,
thereby preventing
specific staining of lipids to be done.
Fat, which reduces osmium tetroxide to osmium
dioxide, is stained black, and may be demonstrated
from the tissue by using chrome-osmium solutions or by
the frozen section method
34. PERIODIC ACID SCHIFF (PAS)
• stains glycogen, mucin,
mucoprotein, glycoprotein,
basement membranes,
capsules and blood vessels,
as well as fungi and
intracellular carbohydrates.
Cells that secrete mucus are
also strongly stained.
• Aldehydes = reddish purple
35. PHOSPHOTUNGSTIC ACIDa
common negative stain for
viruses, nerves, polysaccharides
Result: blue
phosphotungstic acid–hematoxylin (PTAH) staining of
formalin-fixed-paraffin embedded human skeletal muscle
36. PICRIC ACID
• is employed as a contrast
stain to acid fuchsin, for the
demonstration of connective
tissue (Van Gieson's stain) Picric Acid:
Nile Red: picric acid as a differentiator -
Adipocytes • a cytoplasmic stain in contrast the elastic fibres are definitely
crisper
to basic dyes, a counterstain to
crystal violet
• a tissue fixative and decalcifying agent
37. PRUSSIAN BLUE
• an insoluble colored salt of ferric ferrocyanide
normally utilized for the manufacture of paints, but
may be used for microanatomical color contrast of
specimens and for demonstration of the blood and
lymph vessels by injection (intravital staining).
Oil Red O:
Prussian Blue:
Iron staining in
Hemochromatosis
38. RHODAMINE B
• is used with osmic acid to fix and
stain blood and glandular tissues.
39. SAFRANIN (or Safranin O)
• is a nuclear stain
• produces red nuclei, and is
used primarily as counterstain.
• May also be used to give a
yellow color to collagen. Safranin O: Cartilage
40. SILVER NITRATE
• is used in 10% aqueous
solution to prepare various
dilutions to be used in
identification of spirochetes,
reticulum and other fiber stains.
41. TOLUIDINE BLUE
• substitute for thionine in fresh
frozen tissue sections.
PAS staining:
Glycogen in Urothelial CA • It is recommended for staining
of Nissl granules or Toluidine Blue:
Glomerulus
chromophilic bodies.
• nuclei = blue
• used to differentiate different types of granules
• often used as a preliminary stain for electron
microscopy
42. VAN GIESON STAIN It is also fat soluble, and is good as a fat stain for Central
• binds to collagen in the Nervous System tissues
extracellular matrix = pink
• often combined with a stain 3. Sudan IV (Scharlach R) - red
for elastic fibers = black -it has no secondary amino group
-it does not color phospholipids
43. VICTORIA BLUE of the fine lipid droplets.
• is used for demonstration of -Addition of benzoic acid Sudan IV:
Liver Fat cells (Red)
neuroglia in frozen sections. intensifies fat and prevents rapid
deterioration of the solution.
44. VON KOSSA STAIN It is recommended for staining triglycerides (neutral
• a silver reduction method that lipids), giving them a deep and intense red stain.
demonstrates phosphates and
carbonates, but are usually Chief Solvents Used for Stains
present along with calcium 1. WATER
• most useful when large • should always be distilled
amounts are present (bone) Von Kossa: 2. ALCOHOL
Calcium deposit (Black)
• Ethyl alcohol may be used in various
45. WRIGHT STAIN concentrations. Methyl alcohol, if to be used, is
• causes blood cells to usually absolute, it should be acetone free.
exhibit four major 3. ANILINE WATER
staining properties that • 10 ml. of aniline is added to every 1/2 to 1 liter
allow the cell types to be of hot distilled water, shaken, cooled and
distinguished: filtered.
4. PHENOL
• is used in aqueous solution of 0.5 - 5%.
Oil Soluble Dyes (Lysochromes)
Lysochromes (oil soluble dyes) are not real dyes in the
usual sense of the word, because they do not have
auxochrome groups. --------------------- END ---------------------
CheDabuconM.D.
• They give color to lipids, simply because they Bruce-Gregorios, Jocelyn H. (2016). Histopathologic techniques. revised ed. Quezon City
are more soluble in the lipid medium of the : Goodwill Trading. 356 p.: ill. RES/616.07583028/B83/2016.

tissues than in their medium of 70% alcohol.

Oil soluble dyes are available in the following forms
which are used for the demonstration of intracellular
fats:
1. Sudan Black B – black
-most sensitive of the oil
soluble dyes.
-much greater affinity for
phospholipid than other
lysochromes - coloring Sudan Black:
Fat in FFSection
neutral lipids
(triglycerides) by simple dissolution of the dye
-The ability of fats to adsorb Sudan Black is related
to dye concentration, temperature and physical
state of the fats

2. Sudan III – orange

-Sudan III was the first Sudan dye
to be introduced into
Sudan III:
histochemistry. Adipose Cell (Orange)
Staining of Carbohydrates (CHO)
Glycogen – main storage form
Mucin – CHO bound to other substances
-hexosamines that is secreted by the goblet cells
= both stained by PAS (Periodic Acid Shiff)
CHO oxidize the 1,2 glycol group
➔ aldehydes ➔ a red magenta
or purplish-pink
*intensity of PAS reaction is proportional to
the content of sugar
Most fixatives can be used, except those that
contain:
 Osmic acid
 Chromates and;
 Pemanganates
PAS staining can be used to demonstrate the
following:
✓ Polysaccharides
✓ Neutral mucus substances
✓ Tissue basement membranes Fungal
organisms - stains the cell walls contining high
levels of CHO
Staining Reaction:
Carboxyl group (OH) + Periodic Acid (Oxidizing A. )
oxidation Aldehyde + Shiff’s Reagent
➔Magenta Color + Hematoxylin (Counterstain
Results:
• PAS positive substances: Red or Magenta Red
• Nuclei: Blue (Hematoxylin)
NOTES:
✓ Mucoproteins are the most common PAS positive
✓ Carbohydrates, glycoproteins, glycolipids,
unsaturated lipids and phospholipids are also PAS-
positive
✓ PAS-positive staining can also be found in certain
bacteria, fungi, kerasin, connective tissue mucin,
basement membrane, thyroid and cartilage.
Glycogen
• the main storage form of glucose,
manufactured and stored chiefly in the liver,
• It is very soluble in water and insoluble in
alcohol. Theoretically, therefore, alcoholic
solutions are supposed to be the best
fixatives and aqueous fixatives are not
suitable.
a. PAS with Diastase for
Glycogen Demonstration
Results:
• Glycogen: Red
• Nuclei: Blue-black
• CONTROL: Only the nuclei are stained
b. Best's Carmine staining is brought about due to the
affinity of alkaline carminic acid for glycogen.
• This method is selectively and highly specific
for glycogen
Staining Reaction:
Glycogen + Best carmine = Bright Red +
Ehrlich's hematoxylin (CS)
Results:
• Glycogen - Bright Red
• Nuclei - Blue
3. Langhan's Iodine Method (Carleton's Modification)
Langhan's Iodine is the oldest stain, and is
already considered obsolete because it is not
specific for glycogen
Results:
• Glycogen- Mahogany Brown
Mucins
• Polysaccharides bound to other substances
(the nature of which serves as the basis for
their classification), forming the ground
substance of connective tissues primarily.
• Light pink with eosin when stained with H&E
• when Erlich's Hematoxylin is used, acid
mucopoysaccharides esp. from connective
tissue will stain bluish or basophilic
Depending upon the nature of the substance with
which they are bound, mucins may be classified into:
Acid mucopolysaccharides
-with hexuronic acid as a secondary
carbohydrate constituent, bound to sulfuric
acid esters and proteins
-not strongly PAS positive.
a. METACHROMATIC STAINING
• Acid mucin is metachromatic (producing a
color that is different that normally exhibited
by the dye), while neutral mucin is not.
• The most useful
metachromatic dye for
acid mucin is Azure A.
Results:
Connective tissue mucin: Crimson or Red / Violet
Glycosaminoglycans (GAGs): Red Purple
Other tissue components: blue
b. ALCIAN BLUE TECHNIQUE
-the most popular method
Staining Reaction:
Mucin + 3% acetic acid at pH 2.5
Results:
Acid mucins: Blue
Nuclei: Red
c. Combined Alcian Blue-Pas-Hematoxylin Technique • They stain red with PAS but do not stain with
For Acid And Neutral Mucins Alcian blue, colloidal iron, muciramide, or
• useful for demonstrating presence of any metachromatic dyes.
mucins, especially for separating acid mucins
and neutral mucins. PAS-positive carbohydrates:
• Only the neutral mucin will be stained by PAS, 1. MUCOPROTEINS
while acid mucins will be Mucoproteins - contain more than 4% hexosamine
stained by Alcian Blue. and are found in epithelial, glandular, ductal mucins
Results:
Acid mucins: Blue Glycoproteins - on the other hand, contain less than
Neutral mucins: Magenta 4% hexosamine and are found in serum albumin and
Nuclei: Pale blue serum globulin.

d. COLLOIDAL (Dialyzed ) IRON TECHNIQUE 2. GLYCOLIPIDS

This works on the principle that at low pH, colloidal • They are bound to certain lipid constituents
iron will be adsorbed onto tissue and include gangliosides found in the gray
containing acid mucins, and matter of CNS and cerebrosides, found mainly
subsequently visualized by in white matter of the brain, particularly
conversion to ferric ferrocyanide kerasin.
(prussian blue) using the
conventional Perl's Technique. LIPID
• All fat and fat like, or fat
e. GOMORI'S ALDEHYDE FUCHSIN STAIN containing substances.
Aldehyde fuchsin was • All fats have the common
first introduced by Gomori in property of being soluble in
1950 as an elastic tissue stain. organic solvents, and
It employs basic fuchsin plus insolubility in water.
Elastic tissue
aldehyde to demonstrate • Lipids are best demonstrated on cryostat sections
sulfur-containing compounds. of fresh unfixed tissue since there is no really
good fixative available.
f. MUCICARMINE STAIN • Phospholipids and neutral fats will be lost during
Staining Reaction: routine dehydration and embedding unless they
Mucin + aluminum are treated with potassium dichromate or osmic
hydroxide + carmine acid, which are the only agents that truly fix
• One of the organisms that is lipids.
identified using this staining technique • To preserve lipids, polyethylene glycols
is Cryptococcus neoformans (carbowaxes)
They are generally classified into:
g. FLUORESCENT ACRIDINE ORANGE ❖ Simple lipids (neutral fat)
TECHNIQUE • esters of fatty acids with alcohols
Acridine Orange is a fluorescent stain • usually found in the body as energy stores
(fluorochrome) that can be used to in adipose tissue.
demonstrate acid mucins Triglycerides
- Esters of fatty acids with glycerol constituents
- serving as storage fats in animals with high
Neutral mucopolysaccharides solubility for certain non-ionic colored
• These substances contain hexoses as their second substances (lysochromes) stainable by Sudan
carbohydrate component and are found in the Black B, Sudan IV and Oil Red O.
epithelial and intestinal glands and in the
prostate. ❖ Compound lipids
• The combination of the alcian blue and the PAS -Consists of a fatty acid, an alcohol and one or
techniques can be used as a means of more other groups such as Phosphorus or
distinguishing neutral mucins from acid mucins. Nitrogen
- found in central nervous system.
 a. PHOSPHOLIPIDS 2. β-Naphthols such as the original diazo dyes:
Important components of cellular membranes a. Sudan III
particularly found in mitochondria and nervous • predominantly for
tissue elements and are readily stained by Sudan staining TAG mal
Black B and acid hematin. tissues (frozen sections)

b. GLYCOLIPIDS b. Sudan IV (Scharlach R)

Are composed of fatty acids and hexoses, • stains fats with a more
possessing characteristics of both lipids and brilliant red color than
carbohydrates and Sudan III (stains lipids
orange-red)
❖ Derived lipids • the most commonly
-fatty acids that are derived from hydrolysis of used stain for lipid demonstration
simple and compound lipids Results:
- examples are cholesterol, bile acids, sex Lipids(mainly TAG)- Red
hormones and adrenocortical hormones. Nuclei- Black / Blue

Adipose Tissue 3. Oil Red O

• Adipose tissue or fat is • stains neutral fats and lipofuchsin well
distributed throughout • the intensity of its red coloration makes ORO
the body in distinct the preferred choice for detecting the
“white” and “brown” presence of lipids in tissues
adipose tissue deposits. Oil Red O Method in Dextrin
a. White Adipose Tissue (WAT) - composed of Results:
unilocular lipid-filled adipocytes that specialize in lipid Fat- Brilliant Red
storage Nuclei- Blue
b. Brown Adipose Tissue (BAT)- composed of
multilocular adipocytes that specialize in lipid burning
• Fat cells appeare as “signet rings” on H&E stain 4. Osmic Acid Stain for fats
• Osmium tetroxide is not a dye but is an unstable
SUDANOPHILIA oxide which is reduced to a black substance by
• The property of tissues to be stained with fat or unsaturated fats and fatty acids.
oil-soluble dyes, regardless of the type of dye Results:
used, due to their essential lipid nature. Nuclei- Yellow- Orange
• Staining with these dyes is regarded as specific for Fats- Black
lipids, especially for simple lipids (neutral fat).
5. Nile Blue Sulfate Method for Fats
Oil soluble dyes are usually divided into the main • preliminary indicator of the type of lipid
groups: present in the tissue section.
1. Basic Aryl amines with very low water solubility:
a. Sudan Black B • It is a dye capable of differentiating two lipid
• most sensitive and versatile lipid stain known classes simultaneously by the action of its two
• stains Phospholipids as well as neutral fats components:
• does not stain crystalline cholesterol ▪ Red oxazone which
• FFA tend to be dissolve in dissolves neutral lipids.
the alcoholic dye bath ▪ Blue oxazine which is
Results: basic and reacts with
Lipids: Blue Black phospholipids and free
Nuclei: Red fatty acids.

b. Sudan Red VII B

6. Nile red
• an excellent stain that is present as a minor
component of commercial preparations of the
non-fluorescent lipid stain Nile Blue
• it can serve as a sensitive vital stain for the
detection of cytoplasmic lipid droplets.
Neutral lipids: Yellow
Histochemical Methods
• Unlike the fat stains described above,
histochemical methods involve chemical
reactions with specific groups, radicals or bonds
in the lipid molecule.
• Many of these methods are utilized mainly for
research studies
Free Fatty Acids
• Free fatty acids bind heavy metal ions such as
copper to form soaps which can be stained
with Weigert’s lithium hematoxylin,
dimethylaminobenzidine rhodamine, or
rubeanic acid.
Cholesterol
• An enzymatic method for the histochemical
localization of free cholesterol, cholesterol
esters, or both makes it possible to be
compatible with routine histological staining
procedures.
Cerebrosides
• Cerebrosides and related lipids are stained by
PAS method and can be distinguished from
glycogen by removal with diastase.
➢ Toluidine Blue-Acetone Method for Sulfatide
Results:
Sulfatide(sulfate esters of
cerebrosides) stain
metachromatic red-brown
or yellow
PROTEINS (CHON + AA linked by peptide bonds)
• the basic component of living cells
They occur in tissues either as:
Simple CHON
• AA + occasional small CHO compounds
• e.g., albumins, globulins, structural proteins,
enzymes
Conjugated CHON
• Simple CHON + non-CHON material in the
body = complex CHON
• e.g., lipoproteins, mucoproteins and
nucleoproteins
Derived CHON
• derived from simple or conjugated CHON by
physical or chemical means
• e.g., denatured CHON and peptides
NUCLEIC ACIDS
• Nucleic acids are usually
combined with basic
proteins to form
nucleoproteins.
Two major nucleic acids:
• Deoxyribonucleic Acid (DNA)
found mainly in the nucleus of the cell
• Ribonucleic Acid (RNA)
found in the cytoplasm, and a lesser extent
in the nucleus, particularly in the nucleolus
Principles of staining
• Staining depends largely on the attachment of
dyes to proteins that have both positively and
negatively charged groups.
• Phosphate groups are also important in nuclear
staining.
• DNA/RNA in the nucleus, and RNA in ribosomes
in the rER are both acidic because the backbones
of nucleic acids are negatively charged due to the
presence of phosphate groups.
• Therefore, dyes like hematoxylin will bind to DNA
and RNA and stain them violet
Histochemical Identification of Proteins
• Histochemical methods are used to demonstrate
the presence of amino acid molecules rather than
whole protein molecules.
• It is important to avoid fixatives such as mercuric
chloride which react with amino acid groups.
• Neutral buffered formol saline is the most
commonly used fixative for amino acid
histochemistry.
Histochemical Stain for Proteins Results:
Methyl Green is highly
1. Alkaline Fast-Green Method For Basic Proteins
selective for DNA (chromatin) :
• Fast Green is an acid dye that stains basic groups
green or blue green
in the tissues, particularly basic protamines and
Pyronin is specific for RNA
histones.
(nucleoli) : rose-red
Results:
Histones & Protamines (found in
Methyl green-pyronin stain
nuclei) - Green
is also utilized to detect the
presence of plasma cells
2. Peracetic Acid-Alcian Blue For Cystine And CysteinE
and lymphocytes
• Peracetic Acid oxidizes cystine and cysteine,
(cytoplasm): purple
forming strong cysteic acid which is stained
blue-green by a basic dye.
Results:
Cysteine (found in nuclei) –
FLUORESCENT STAINING FOR DNA AND RNA
Blue Green Fluorochromes are fluorescent dyes which emit light
or visible radiation energy when excited by light of
3. Alcian Blue-Pas Staining For Proteoglycans shorter wavelength, visible (380-700nm) or ultraviolet.
Proteoglycans (glycoprotein/GAG) - these are
proteins that are heavily Several fluorochromes are presently available:
glycosylated, negatively 1. Fluorescein
charged under physiological • The most widely used fluorochrome, because
conditions due to the of its wide absorption spectrum and blue light
occurrence of sulfate and range.
uronic acid groups • Its characteristic apple
green emission is rarely
Result: seen as "autofluorescence"
Acid mucins : blue in mammalian tissue,
Neutral mucin/Proteoglycan: Magenta Red which is often blue in
color.
Staining of Nucleic Acids 2. Rhodamine
• Demonstration of nucleic acids depends upon Conjugates absorb maximally in
either reaction of dyes with the phosphate green light, exhibiting an
groups, or production of aldehydes from the sugar orange-red emission and are
(deoxyribose). commonly used in two-color
techniques
DNA is Demonstrated by the Feulgen Technique which 3. Acridine Orange
uses 1 M HCl at 60°C to hydrolyze and break the The most commonly used fluorochrome to
purine-deoxyribose bond, thereby exposing the demonstrate DNA and RNA in fresh or fixed
aldehydes which are then stained by Schiff's reagent. tissues. This is used for screening of cervical
• Feulgen's reaction is the most reliable and smears for cancer cells.
specific histochemical staining technique for DNA, Results:
best known for chromatin and nucleoproteins. DNA: Yellow green
Result: RNA: Brick to orange red
DNA: Red-purple
Cytoplasm: Green 4. Acriflavine
As a 0.01% alcoholic solution,
can be used as an alternative to
basic fuchsin in Schiff's reagent,
for the Feulgen technique of
RNA is demonstrated by the Methyl Green-Pyronin acid hydrolysis.
Technique where methyl green stains the nuclei by DNA is stained by a fluorescent
binding preferentially and specifically to DNA, while yellow color in this Feulgen-type
pyronin binds to RNA and stains the cytoplasm red. reaction.
IMMUNOHISTOCHEMISTRY for CHON
• most commonly applied protein immunostaining
technique.
IN-SITU HYBRIDIZATION for CHON
• the definitive, most sensitive technique for
identifying DNA
It has become applicable in routine laboratories due
to the introduction of:
a. Recombinant DNA technology
By which sequences of nucleic acids, either DNA or
RNA, could be amplified by cloning and purified to
very high levels
b. Nick translation methods
Incorporating labeled nucleotides into purified DNA
and synthesizing a biotinylated nucleotide for non-
radioactive in-situ labeling.
In-situ hybridization uses a labeled complementary
DNA, RNA or modified nucleic acid strands (probes) to
localize specific DNA, RNA, nucleic acid sequence or
gene expression within a cell, in contrast with
immunohistochemistry, which usually localizes
proteins in tissue sections.
POLYMERASE CHAIN REACTION (PCR)
- nucleic acid primers are hybridized to a
target nucleic acid sequence to allow the
enzymatic copying and subsequent
amplification of the sequence in question
ENZYME HISTOCHEMISTRY
• serves to detect early metabolic changes in
biopsy and autopsy tissue before manifestation
on H&E.
ENZYMES
• tissue components that serve as catalysts for most
biological reactions.
They may be:
✓ Bound to specific cell components e.g.,
mitochondrial enzymes
✓ Free and soluble in the cytoplasm and body
fluids.
• With a few exceptions (e.g., chloroacetate
esterase), frozen sections are generally required
for histochemical demonstration of enzymes.
－ Tissues frozen to -70°C or below are usually well
preserved, with little loss of enzyme activity.
• In general, the best fixative for all enzymes is
chilled acetone, which, of all fixatives, causes the
least inactivation.
Histochemical Demonstration of Enzymes:
a. Metal Precipitation
the most common technique for histochemical
demonstration of enzymes, based on
simultaneous capture or coupling, which involves
the use of suitable substrate and diazonium salt.
b. The use soluble substrates, which undergo
molecular rearrangement to give a colored
insoluble reaction product after enzyme
hydrolysis.
Enzymes for which histochemical techniques are
known belong in one of the two groups:
a. Oxidative enzymes
• they catalyze the reaction between substrate
an atmospheric oxygen
b. Hydrolytic enzyme
• they are complex catalytic proteins that use
water to break down molecules (CHON, CHO,
Nucleic acids, starch, fat, phosphate esters,
etc.) into their simplest units
• Most of the histochemically demonstrable
hydrolytic enzymes belong to the group of
esterase which hydrolyze esteric linkages.
CONNECTIVE TISSUE
Constitutes a non-living framework within
the various organs, and is made up of various cell
components that are found in between other tissues
everywhere in the body.
Connective tissue matrix has a ground
substance consisting of water that is stabilized by
fibers stabilized by GAGs, proteoglycans, and
glycoproteins
The most common connective tissue cells:
a. Fibroblasts - secrete collagen and other elements
of the ECM
b. Adipocytes - mesenchymal cells which store fat
c. Mast cells, macrophages, lymphocytes - cells with
immune function
1. Loose (Areolar) Connective Tissue
• Connects the epithelial surfaces
to the underlying structures and
permits free passage of
nutrients.
2. Adipose Tissue
• Derived from areolar tissue,
made up mostly of fat cells
surrounded by a well-developed
network of reticular fibers.
3. Dense Connective Tissue c. Mallory's Aniline Blue Stain
• Made up of organized, dense • This is an excellent and colorful method of
masses of collagenous fibers demonstrating connective tissue fibers.
and fibroblasts that are oriented • It is often used to differentiate acidophilic
in the same direction, as seen in extracellular fibers from acidophilic cytoplasm.
the tendons and ligament Results:
4. Cartilage Nuclei, fibrin, muscle fibers: Red
• A fairly dense network of Collagen: Blue
collagenous fibers encased in or Cartilage, bone, mucus: shades of blue
mixed with an amorphous or Blood, myelin: Yellow
homogenous intercellular Elastic fibers: Pale pink/yellow or unstained
substance of chondroitin sulfate.
5. Reticulin (Reticular Connective Tissue)
• made up of extracellular
delicate fine branching fibers
that are not visible in routine
H&E staining method.
• The reticulin fibers are d. Azocarmine Stain
argyrophylic and therefore are best stained by • This is Heidenhain's modification of Mallory's
silver impregnation technique ➔ black aniline blue stain, using azocarmine dye for
a. Gomori's Silver Impregnation Stain counterstaining.
Result: • It is a valuable stain showing minute details of
Reticulin fibers: Black connective tissue and of renal glomerular
basement membrane as well.
b. GORDON AND SWEET'S METHOD Results:
Result: Amyloid CT, mucous: Deep blue
Reticulin fibers: Black Nuclei: Red
Nuclei: Black
Background: Red
6. Elastic Tissue
6. Collagen • they are present in the skin,
• Collagen forms a coarser extracellular framework ligaments, aorta, arterial
than reticulin. elastic lamina, and the lungs
• The most commonly used acid is picric acid, which • elastic fibers are composed
also counterstain for muscle and cytoplasm. of the protein elastin that
a. Van Gieson's Stain are highly distensible and, when broken, recoil
• The simplest method of differential staining like rubber bands
of collagen using a mixture of picric acid and • Elastic tissue is isotropic and gives a yellowish
acid fuchsin fluorescence in UV light (blue if unstained).
Results: • In the presence of ferric salts (oxidizers) elastic
Nuclei: Brownish black to black fibers stain with basic fuchsin.
Collagen (fibrous connective • From acidic solutions, the fibers are stained
tissue): Pink or deep red selectively by the weak acid orcein.
Muscle, Cytoplasm , RBC and
Fibrin: Yellow a. Weigert's Resorcin-Fuchsin Elastic Tissue Stain
b. Masson's Trichrome Stain Result:
• Uses dyes in acid solution involving nuclear Elastic fibers:
staining with iron hematoxylin, followed by Brown to
cytoplasmic staining with a red dye (e.g. Ponceau purple/blue-black
phosphotungstic acid with Methyl violet
Results: Other substances:
Muscles, RBC and Keratin: Red depending on the
Nuclei: Blue-black counterstain used
Collagen & mucus: Blue Nuclei: Red
b. Orcein (Taenzer-Unna Orcein Method) b. The Periodic Acid Schiff (PAS) technique
• Orcein is a naturally occurring vegetable dye, • Is the common method
which has now been synthesized. It is used to used for demonstration of
stain elastic fibers, especially in dermatology due basement membrane,
to demonstration of the finest and most delicate particularly the glomerular
fibers found in the skin. basement membrane of
Result: the kidney, due to its
Elastic fibers: Dark brown carbohydrate content
Nuclei: Blue
7. Blood Plasma
c. Verhoeff's Stain • the watery component
(Verhoeff-van Giesson stain) that serves as the matrix
• most commonly used because it is quick, and of blood containing
produces intense staining of elastic fibers that are many dissolved
not easily distinguished by H&E staining. substances, such as
Result: protein, glucose, mineral
Elastic fibers: Black ions, hormones, carbon dioxide, etc.
Nuclei: Gray to black
Collagen: Red
Cytoplasm and muscle: Yellow PATHOLOGIC CHANGES AND DEPOSITS FOUND IN
CONNECTIVE TISSUES
d. Gomori's Aldehyde-Fuchsin Stain 1. FIBRIN
• HCl and paraformaldehyde are added to an • It is an insoluble fibrillar protein material derived
alcoholic solution of basic fuchsin to form from the fibrinogen in blood plasma.
aldehyde fuchsin. Schiff bases are formed by the • It is commonly seen after tissue damage, in blood
aldehyde and the fuchsin, clots and in acute inflammatory reactions
and staining is intensified by • During clotting, hemoglobin and glycoproteins
prior oxidation. become entangled with the fibri giving a weak
Result: reaction when tested for hemoglobin and
Elastic fibers: Deep blue to glycoproteins.
purple • It is an acidophil material, that is, it is stained by
Other tissue elements: Green acid dyes and can often be seen in H&E stain as
distinctly pink but less brilliant than the
e. Krajian's Technique erythrocytes.
• A rapid method for
staining elastic fibers, a. Malory's Phophotungsic Acid Hematoxylin (Ptah)
fibrin and amyloid, Method
employing Congo red. • The PTAH staining method relies on acid-base
Result: chemistry to stain collagen and muscle fibers.
Elastic fibers: Bright red • This stain has been referred to as a polychrome
Fibrin and connective tissues: Dark blue stain because one solution gives two major colors.
RBCs: Orange-Yellow Results:
7. Basement membranes Fibrin, muscle striations,
-are found throughout the body as a resilient neuroglia, amoeba: Dark
matrix made up of carbohydrate complexes. blue
a. Jones' Impregnation Technique (Silver Nuclei, cilia, RBCs: Blue
Methenamine) Myelin: Lighter blue
- excellent or the demonstration of Collagen, osteoid,
glomerular and tubular basement cartilage, elastic fibers: Deep brownish red
membranes of kidney biopsies Cytoplasm: Pale pinkish brown
Results:
Basement membranes: Black
Nuclei: Blue
Background: Pink
2. FIBRINOID Fluorescence may be
• Fibrinoid is an eosinophilic imparted to amyloid by
material that has identical staining with Thioflavine T,
staining reactions to fibrin, and exposing the tissue to
but occurs in tissues under UV or Quartz iodine
different conditions and lamps, emitting short
disorders. wavelength blue light.
• It is a mixture of exudate and altered cytoplasmic
constituents, forming a homogenous eosinophilic
material which gives the same staining reaction
as fibrin.
3. HYALIN -------- END ------
c h e d a b u c o n M D
• appearing in the form of a
Bruce-Gregorios, Jocelyn H. (2016). Histopathologic techniques. revised ed. Quezon
smooth homogenous and City : Goodwill Trading. 356 p.: ill. RES/616.07583028/B83/2016.
acellular eosinophilic
proteinaceous material in
routine H& E stains
particularly in degenerated collagen,
hypertension, atheroma and diabetic kidney.
4. AMYLOID
• a semi-translucent groundglass or hyaline
eosinophilic substance made up of a chondroitin
sulfuric acid-protein complex, deposited in
connective tissue cells and other organs
• It is stained by acid dyes and is pale pink in H&Et
is largely protein, but may contain other materials.

a. Gram's Iodine Stain

• The purple staining of amyloid with iodine
solutions, changing to a blue color on addition of
weak sulfuric acid, is the oldest staining method
For Identifying Amyloid Deposits In The Tissue.
B. Congo Red Method
• Congo red gives amyloid a deep pink to red
color with subsequent green birefringence
under polarizing lens.
• When viewed with polarized light under high
field using a polarizing filter, congo red-
stained amyloid exhibits a characteristic bright
"apple green" birefringence.

3. Induced Fluorescence Staining With Thioflavine

Fluorescence dye staining is a very sensitive technique
for demonstrating various materials, including
amyloid.