RAMOS, Jerome S.
04/26/18
MERRERA, Myles Janine T.
MONTANIEL, Princess G.
MUÑOZ, Marian Louie G.
PINEDA, Eunice
1. Gomori’s Silver Impregnation - To stain reticulin, a fibrillary
stain
2. Van Gieson’s stain
3. Masson’s trichrome stain
4. Mallory’s aniline blue stain
5. Azocarmine stain
6. Welgert’s elastic tissue stain
7. Verhoeff’s elastic method
8. Orcein (Taenzer-Unna Orcein
method)
Staining Techniques
CONNECTIVE TISSUE
PURPOSE
extracellular matrix found throughout the
tissues of the body
- Simplest method of differential staining
of collagen using a mixture of picric acid
and acid fuchsin
- Uses dyes in acid solution involving
nuclear staining with iron hematoxylin,
followed by cytoplasmic staining with a
red dye
- Used to stain collagen fibers, reticulum,
hyaline fibrils, fibroglia fibrils, smooth &
striated muscle fibers and amyloid
- Excellent & colorful method of
demonstrating connective tissue fibers
- Heidenhain’s modification of Mallory’s
aniline blue stain, use azocarmine dye for
staining
- Valuable stain showing minute details of
connective tissue and of renal glomerular
basement membrane
- Used to stain elastic fibers present in
the skin, ligaments, aorta, arterial elastic
lamina, and lung
- Used to stain elastic fibers present in
the skin, ligaments, aorta, arterial elastic
lamina, and lung
- Used to stain elastic fibers, especially in
dermatology due to demonstration of the
9:30-10:30 TTH
RESULT OF STAINING
 Reticulin fibers: Black
 Nuclei: Brownish black
 Collagen (fibrous connective tissue): Pink, or
Deep red
 Musce, Cytoplasm, RBC & Fibrin: Yellow
 Muscle, RBC, & Keratin: Red
 Nuclei: Blue-black
 Collagen & Mucus: Blue
 Collagen fibrils, cytoplasm, fibroglia fibrils,
axon cylinders & neuroglia fibers: Red
 Elastic fibers: Pale pink or yellow
 RBC & myelin sheets: Yellow
If it is desired to bring out collagen fibrils as
sharply as possible, omit the staining with acid
fuchsin:
 Nuclei & cytoplasm: Yellow
 Reticulum: Stand out more prominently
 Amyloid connective tissues & mucous colloid:
Deep blue
 Nuclei: Red
 Elastic fibers: Dark-blue or blue-black on a
clear background
(Other structures are colored depending on the
counterstain used)
 Nuclei: Red with carmine before or after
staining of fibers
 Elastic fibers: Black
 Nuclei: Gray to black
 Collagen: Red
 Cytoplasm & muscle: Yellow
 Elastic fiber: Dark brown
 Nuclei: Blue

9. Krajian’s technique
finest and most delicate fibers found in
the skin
- Rapid method for staining elastic fibers,
fibrin, and amyloid employing congo red
 Elastic fibers: Bright red
 Fibrin & connective tissues: Dark blue
 RBC: Orange-yellow

10. MSB technique
- Used to stain fibrin, an insoluble fibrillar  Nuclei: Blue
protein resulting from polymerization or  Erythrocytes: Yellow
enzymatic coagulation of plasma globulin  Muscle: Red
and fibrinogen, forming bundles which  Collagen: Blue
contract into dense homogenous masses  Fibrin: Red
by employing martius yellow C1 10315, (Early fibrin may stain yellow, and very old fibrin
brilliant crystal scarlet C1 16250 and may stain blue)
soluble blue C1 42780

CARBOHYDRATES

PURPOSE RESULT OF STAINING

1. PAS with diastase - PAS Technique with diastase control is  Nuclei: blue
the method of choice for glycogen  Glycogen: red
staining

2. Best carmine method
3. Langhan’s iodine method
(Carleston’s Modification)
- This method is selective but not as
highly specific for glycogen as the PAS
method with and without diastase. Mast
cell granules, fibrin and mucin are also
stained, albeit weakly
- Mast cell granules, fibrin and mucin are
also stained, albeit weakly
 Nuclei blue or grayish blue
 Glycogen: pink to red
 Mucin: weak red
 Nuclei blue or grayish blue
 Glycogen: pink to red

4. Uranyl Acetate Azure A - To stain acid mucopolysaccharides  Mucopolysaccharide red purple

metachromatic technique
5. Fresh frozen azure A
metachromatic staining
6. Metachromatic toluidine
blue staining
7. Alcian blue technique
8. Combined alcian blue PAS
technique
Tissue background blue
- To stain glycosaminoglycan  Glycosaminoglycan- red purple
- Toluidine blue is a basic thiazine  Mucopolysaccharide red purple
metachromatic dye with high affinity for  Tissue background blue
acidic tissue components and nucleic
acids
. It is also used to highlight tissue
components such as cartilage or certain
types of mucin.
- Alcian blue is a histological dye that  Acid mucins blue
forms electrostatic bonds with certain  Nuclei red
tissue components containing either
carboxyl or sulfate groups (such as
sulfated or acid mucins which are
specifically and intensely stained.
- Alcian blue is the most popular method
for general demonstration of acid
mucopolysaccharides, using 3% acetic
acid at pH 2.5.
- This combined technique is useful for  Acid mucins blue
demonstrating the presence of any  Neutral mucins magenta

9. Gomori aldehyde fuchsin
stain
10. Muricarmine stain
1. Modified gomori’s trichrome
stain
2. Rapid gomori trichrome stain
3. Mallory’s phophotungstic
acid hematoxylin
4.Heidenhan’s iron hematoxylin
method
5. Lissamine test red tartrazine
method
6. Schmorl’s picrothionin - Demonstration of thionin precipitate
method
7. Ground section preparation
mucin, especially for separating acid
mucins and neutral mucins.
- A number of other tissue components
are equally stained, including
acid mucopoly-saccharides, sulfated
mucosubstances, pancreatic islets of
Langerhans, thyrotrophic hormones, and
secretory substances.
- It is also used to
stain mast cells, particularly when no
counterstaining is done.
- Its large molecular size also allows the
dye complex to penetrate and bind to
acidic mucins but not to other acidic
substances such as nucleic acids.
MUSCLE AND BONE
PURPOSE
-Demonstrate frozen sections in muscle
biopsy
-Detects ragged fibers in mitochondrial
myopathy
-Demonstrate only muscle biopsies and is
for staining of freshly cut cryostat sections
mounted on coverslips
- Ideal for demonstrating striated muscle
fibers and mitochondria, often without a
counterstain
-It is used to identify contraction bands, as
seen in contraction band necrosis
-Method recommended for monochrome
photography
- General stain for epon-embedded tissues
as well as paraffin and celloidin-
embedded tissues
- Routine staining method for bone
histology (muscle striations are well-
shown)
within the lacunae and bone canaliculi
- Used for demonstration of lacunae and
canaliculi in bone
 Sulfated mucins- purple
 Carboxylate forms - blue
 Mucins red
 Nuclei blue
 Background unstained
RESULT OF STAINING
 Muscle fibers – Green
 Ragged areas - Red
 Myofibers and connective tissue – different
shades of Green
 Nuclei – Red purple
 Intermyofibrillary network - Red
 Myofibrils – Green
 Intermyofibrillar material - Bright red
 Nemaline rods, ragged red fibers - Red on a
blue/green background.
 Muscle, neuroglia – Blue
 Nuclei - Deep blue
 Cytoplasm - Pale pinkish brown
 Fibrin - Blue
 Collagen - Deep brownish red
 Coarse elastic fibers - Bluish
 Myelin - Blue to blue-gray
 Muscle striations, mitochondria, myelin,
and chromatin are stained grey - black
 Nuclei – Black
 Muscles, RBC – Red
 Collagen - Yellow
 Lacunae and canaliculi - Dark brown-black
 Bone matrix - Yellow or brownish-yellow
 Cells - Red
 Lacunae and canaliculi filled with air
lamellae - Brown or brownish black lines

1. Sudan black method
2. Sudan IV
3. Oil red O method in dextrin
4. Osmic acid stain
5. Nile blue sulfate method
6. Toluidine blue acetone
method for sulfatide
1. Perl’s Prussian Blue Method
For Hemosiderin
2. Gomori’s Prussian Blue Stain
For Iron
3. Turnbull’s Blue Reaction For
Ferrous Iron
FAT AND LIPID STAINS
PURPOSE RESULT OF STAINING
- For staining unfixed cryostat sections
preferred, or frozen sections post-fixed in
 Lipids blue - Black
formol calcium
 Nuclei – Red
- For demonstration of neutral
triglycerides and lipids
 Lipids (mainly triglycerides) - Red
- Demonstrates lipids in frozen sections
 Nuclei - Blue/Black
- Stains lipids in fresh frozen or frozen  Lipid – Red
sections post-fixed in neutral buffered  Nuclei - Blue
formalin
- For demonstration of unsaturated fats  Nuclei - Yellow-orange
and staining of cryostat sections  Fats - Black
- Acts as a preliminary indicator of the type  Neutral lipids - Red
of lipid present in the tissue section  Phospholipids and free fatty acids - Blue
- It serves as a sensitive vital stain for the
detection of cytoplasmic lipid droplets
- Demonstrates sulfatide deposits in brain  Sulfatide deposits – Metachromatic red –
and other organs of patients with sulfatide brown or yellow
storage disease
TISSUE PIGMENTS AND DEPOSITS
PURPOSE RESULT OF STAINING
- Perl’s Prussian Blue Method is used to  Hemosiderin and Ferric salts stain deep
demonstrate hemosiderin and is the blue
major stain use to detect iron in the liver.  Other pigments retain their natural color
This histochemical method is based on the  Tissue and Nuclei stain red (accdg to
unmasking of ferric iron by dilute counterstain)
hydrochloric acid which is a component of
the acid ferrocyanide solution.
- The Ferric ion then reacts with dilute
potassium ferrocyanide solution to
produce an insoluble blue compound,
ferric ferrocyanide (Prussian Blue)
- Gomori’s Prussian blue Stain is used to  Iron pigments stain bright blue
detect loosely bound ferric iron in tissue  Nuclei stain Red
sections. This histochemical reaction is  Cytoplasm stain pink to rose
sensitive enough to demonstrate even
minute amounts of iron deposits in bood
cells bone marrow and spleen.
- Turnbull’s blue is rarely used in routine  Hemosiderin stain blue
histology since it is used to detect ferrous  Nuclei stain red
iron which is less commonly found than
ferric iron. The reaction depends upon the
union of ferrous iron with potassium
ferricyanide to form a blue precipitate of
complex ferrous ferricyanide. Ferrous

4. Benzidine Nitroprusside
Stain For Hemoglobin And
Oxidase Granules
5. Staining For Bile Pigments
and Hematoidin and Modified
Fouchet’s Technique
6. Gmelin Technique For Bile
And Hematoidin
salts are stained blue while others remain
unstained.
- Benzidine Nitroprusside demonstrates  Hemoglobin and some “oxidase” granules
hemoglobin as well as the oxidase in Leukocyte stain dark blue
granules. The stain demonstrates the  Nuclei stain red
enzyme hemoglobin peroxidase  Most other tissue components are faint
(pseudoperoxidase activity) which is pink
reasonably stable and withstands short
fixation and paraffin processing.
- Bile pigments contain both conjugated  Bile pigments stain emerald to blue
and unconjugated bilirubin, biliverdin and  Muscles stain yellow
hematoidin, all of which are chemically  Collagen stain red
distinct and show different physical  **Hematoidin is not likely to show any
properties. The most common method for color change with this method
demonstration of bile pigments is the
Modified Fouchet’s Technique in which
the pigment is converted to green color of
biliverdin and blue cholecyanin by te
oxidative reaction of ferric chloride in the
presence of trichloroacetic acid.
- This method shows an identical result  Bile pigments appear as yellow brown
with liver, bile, gallbladder bile and globules
hematoidin. Gmelin’s test if positive, is a  Color spectrum from red to purple to green
diagnostic for bile pigments, although
results produced are only temporary.

7. Schmorls’s Ferric
Ferricyanide Method For
Reducing Substances
- A test to stain for reducing substances in
tissues including melanin, argentaffin
granules, thyroid colloid, keratin,
keratohyalin, and lipofuscin pigments.
Ferricyanide is converted to insoluble
Prussian Blue in the presence of ferric ions
 Bile, Lipofuschins and Melanin stain dark
blue
 Argentaffin cells and chromaffin stain dark
blue
 Thyroid colloid stain dark blue
 Nuclei stain red

8. Gomori’s Aldehyde Fuchsin
Technique For Lipofuchsin
- Gomori’s Aldehyde Fuchsin Technique is  Lipofuchsin stain purple
for demonstration of Liofuchcin which  Background is yellow
may be seen in neurosecretory cells.
Aldehyde fuchsin is thought to be formed
by condensation of aldehyde groups with
free amino groups of basic fuchsin.

9. Mallory’s Fuschin Stain
- Mallory’s Fuschin Stain is used for  Nuclei stain blue
demonstration of hemofuscin.  Hemofuchsin stain red
Hemofuscin has affinity for fuchsin dyes  Hemosiderin is unstained
relatively resistant to decolorization with
alcohol after staining. This stain may be
combined with Prussian blue reaction to
give a complete pigment picture in
hemochromatosis.

10. Masson Fontana Technique
- Masson Fontana Technique is used for  Melanin stain black
routine demonstration of Melanin. This is  Argentaffin cell granules stain black
based on Melanin’s intrinsic property of  Nuclei stain red
reducing ammoniacal silver solutions
without the use of extraneous reducer.
This property is known as “argentaffin
reaction”
 HISTOCHEMISTRY AND OTHER MISCELLANEOUS
1. Enhanced Polymer One Step
Staining
2. Peroxidase – anti-peroxidase
(PAP) Technique
3. Avian Biotin Complex (ABC)
Technique
4. Direct Immunofluoresecenxe
Technique for Solid Tissue
Biopsies
5. Indirect
Immunofluorescence
Technique
6. Frozen Section
Immunocytochemistry
7. Frozen Section
Immunofluorescence
8. DIrect Immunoperoxidase
Method
9. Paraffin Wax Section
Immunoperoxidase Technique
REFERENCES:
Bancroft J.D (2008). Theory and Practice of Histological Techniques. Elsevier Health Sciences
Bruce-Gregorios J.H. (2017). HISTOPATHOLOGIC TECHNIQUES. Published by: Jocelyn H. Brucce-Gregorios,MD, U.S.
Edition
PURPOSE
- To conjugate the primary antibody
directly to the label
- Recommended for use on blood and
bone marrow smears
- To detect antigen or antibody in tissues
-Direct reaction with a fluorescein-
conjugated antibody specific for the
material being sought within the tissue
- Detection of autoantibodies in the
patient’s serum, including the anti-
nuclear antibody, antimitochondrial
antibody, and liver-kidney microsomal
kidney
- To identify antigens in fresh frozen
sections
- To detect antibodies, particularly for the
diagnosis of glomerular diseases in frozen
sections of renal biopsies
- used for the demonstration of various
substances in tissue sections and utilises
labelled or unlabelled antibodies and the
very stable enzyme, horseradish
peroxidase.
- Used as a diagnostic procedure for
formalin-fixed, paraffin-embedded
specimen, and immunolabeling that can
be correlated with morphology
RESULT OF STAINING
 mixed colours, which were distinct and
readily discriminated from red and blue
 Stable, dark brown reaction end product
when antigen is present in the tissue
 greater concentration of enzyme at the
antigenic site and therefore an increase in
signal intensity and sensitivity upon
addition of substrate
 Apple-green fluorescence when fluorescein
is used as a fluorochrome.
 Orange-red fluorescence with rhodamine
conjugates.
 Fluorescence of the target antigen
 Bright yellow fluorescence of target cells
 Bright yellow fluorescence of target cells
 The result can be examined under light
microscope
 The result can be examined under light
microscope