SPECIAL STAINING

VAN GIESON METHOD

Aim : Staining of the connective tissue
Principle:
In the routine staining method collagen, elastic fibers and
smooth muscle appear pink or reddish in colour.
In the Van Gieson stain, collagen and most reticulin stain
selectively with acid aniline dyes (acid fuchsin). Picric acid
acts as counter stain for muscle and cytoplasm and form
complex with the dyes. This complex has special
Affinity for collagen.
Reagents
1. Solution A
(a) Haematoxylin 1.0 gm
(b) Alcohol 95% 100 ml
2. Solution B
(a) 29% (w/v) ferric chloride 4 ml
(b) Conc. Hydrochloric acid 1.0 ml
(c) Distilled water 95.0 ml
3. Weight's iron heamatoxylin solution:
mix equal quantities of solution A
and solution B. Colour of this reagent should appear violet
black.
4. Van Gieson’s solution
(a) Saturated aqueous picric acid – 10 ml
(b) 1% (m/v) acid fuchsin – 1.5ml
It should be freshly prepared
Procedure
(1) Deparaffinize with xylene
(2) Hydration take sections to water
(3) Stain with Weigert’s haematoxylin for 20-40 minutes
(4) Wash in distilled water
(5) Van Gieson stain 1-3 min
(6) Rinse well in distilled water
(7) Dehydrate in absolute alcohol (2 changes)
(8) Clear in xylene (2 changes) (9) Mount in DPX

Results
1. Collagen Red
2. Muscle and Cornified epithelium Yellow
(B) Nuclei Blue to Black
 McMANUS FOR GLYCOGEN (PAS)
Aim – Staining and identification of the various types of
carbohydrates (polysaccharides & mucopolysaccharides).
Principle :
Tissue structures like liver & heart, striated muscles are
studied by Periodic acid Shiff stain. Periodic acid reacts with
aldehyde group of the carbohydrates and afterwards reaction
with the schiff’s reagent produces a red or purple red colour.
Reagents
1. 0.5% w/v periodic acid solution
2. Schiff’s reagent
3. 1 N Hydrochloric acid
4. 0.1 gm of light green 0.1% (v/v) acetic acid.
5. Harris haematoxylin stain
Procedure
1. Deparaffinize and hydrate to distilled water.
2. Oxidize in periodic acid solution for 5 minutes
3. Rinse in distilled water
4. Schiff’s regent solution for 15 minutes.
5. Wash in running water for 10 minutes for pink colour to
develop.
6. Harris haematoxylin for 6 minutes or light green
counter stain for a few seconds.
7. Wash in running water.
8. Differentiate in 1% acid alcohol solution 3-10 quick dips.
9. Wash in running water.
10. Dip in ammonia water to blue the sections
11. Wash in running water for 10 minutes
12. Dehydrate in 95% alcohol, absolute alcohol, clear in
xylene two changes each.
13. Mount in DPX.
Results
With hematoxylin counterstain
1. Nuclei – blue
2. Glycogen, – purple red
3. Fungi – Red
 MAYER’S MUCICARMINE METHOD
Aim: To demonstrate mucin in a tumor or epithelium
 Giemsa stain
Principle:
Giemsa stain is used for nuclear material visualization. The stain is used
to differentiate nuclear material and cytoplasmic material. The
cytoplasm of bacteria possessing a strong affinity for nuclear stain gets
hydrolyzed with hydrochloric acid. Finally, it is stained with Giemsa stain
which differentiates nuclear material and cytoplasmic material.
Procedure:
RESULTS
Bacteria Blue
Nuclei Blue
Cytoplasm of leukocytes May be shades of pink, grey, or blue,
depending on cell type and development

ALCIAN BLUE
Purpose:
Determining the site of a primary tumor in that finding mucin
positive tumor cells in an area that does not contain mucin
producing cells would indicate the tumor did not arise from that
are

PRINCIPLE:
Alcian blue is a group of polyvalent basic dyes that are water soluble.
The blue color is due to the presence of copper in the molecule .The 3%
acetic acid solution (pH2.5), Alcian blue stains both sulfated and
carboxylated acid mucopolysaccharides and sulfated and carboxylate
(glycoproteins). It is believed to form salt linkages with the
Acid groups of acid mucopolysaccharides.

PROCEDURE:
1. Hydrate slides to distilled water.
2. 3% acetic acid, 3 minutes.
3. *Alcian blue solution, microwave: Hi power, 30 seconds.
4. Wash in running water for 2 minutes, rinse in distilled.
5. Nuclear-fast red, 5 minutes, wash in tap water.
6. Dehydrate, clear, and coverslip.
RESULTS:
Acid mucins /mucosubstances: blue
Nuclei (using Nuclear fast red) : reddish pink

Toluidine Blue Staining Protocol for Mast Cells

Purpose:
Demonstrate {Mast cells are found in the connective tissue and their
cytoplasm contains granules (metachromatic) composed of heparin and
histamine].

Principle:
Toluidine blue should stain mast cells red-purple (metachromatic
staining) and the background blue (orthochromatic staining).
Metachromasia, tissue elements staining a different color from the dye
solution, is due to the pH, dye concentration and temperature of the
basic dye. Blue or violet dyes will show a red color shift, and red dyes
will show a yellow color shift with metachromatic tissue elements.

Procedure:
1. Deparaffinize and hydrate sections to distilled water.
2. Stain sections in toluidine blue working solution for 2-3 minutes.
3. Wash in distilled water, 3 changes
4. Dehydrate quickly through 95% and 2 changes of 100% alcohol (10
dips each since stain fades quickly in alcohol).
5. Clear in xylene or xylene substitute, 2 changes, 3 minutes each.
6. Coverslip with resinous mounting medium.

Results:
Mast cells --------------------------- violet/red purple.
Background ------------------------- blue.

MALLORY TRICHROME

PRINCIPLE
In this method, three different dyes are used: carbol fuchsin for nuclear
staining, orange G for cytoplasm and aniline blue for a selective collagen
staining. Selectivity in this procedure is due to different degrees of
affinity between dyes and tissue macromolecules.
A central role is played by phosphomolibdic acid, which acts as a bound
between tissue structures (collagen fibrils, cell membranes), and aniline
blue (amphoteric dye).
Orange G, which is the second component in Mallory’s polychrome
solution, has no affinity to phosphomolibdic acid and is thus used to
stain all remaining structures unbound to phosphotungstic acid.

METHOD
1. Bring sections to water via xylene and ethanol.
2. Place into solution A for 2 minute.
 3. Rinse with distilled water.
4. Place into solution B for 2 minutes.
5. Rinse quickly with distilled water.
6. Place into solution C for 15 minutes.
7. Wash well with distilled water.
8. Dehydrate and differentiate with ethanol.
9. Clear with xylene and mount with a resinous
medium.
Result:
Nuclei or muscle; red
R.b.c orange
Collagen blue

Masson's Trichrome
PURPOSE: Used to differentiate between collagen and
smooth muscle in tumors, and the increase of collagen in
diseases such as cirrhosis. Routine stain for liver and kidney
biopsies
.
Procedure;
1. Bring sections to water via xylene and ethanol.
2. Stain nuclei with Weigert’s iron hematoxylin or equivalent.
3. Wash well in tap water, rinse with distilled water.
4. Place into solution A for 10 minutes.
5. Rinse with distilled water.
6. Place into solution B for 5 minutes.
7. Rinse with distilled water.
8. Place into solution C for 10 minutes.
9. Rinse well with distilled water.
10. Dehydrate with Ethanol.
11. Clear with xylene and mount with a resinous medium.
Results
Cytoplasm – red
Erythrocytes – red
Muscle – red
Collagen – green
Nuclei – dark brown