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Masson's Trichrome Staining Protocol for Collagen Fibers
Description: This method is used for the detection of collagen fibers in tissues such as skin, heart, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The collagen fibers will be stained blue and the nuclei will be stained black and the background is stained red.
Fixation: 10% formalin or Bouin's solution
Section:
paraffin sections at 5 um.
Solutions and Reagents:
Bouin's Solution:
Picric acid (saturated) ------------------- 75 ml
Formaldehyde (37-40%) ------------------ 25 ml
Glacial acetic acid
------------------------- 5 ml
Mix well. This solution will improve Masson Trichrome staining quality.
Weigert's Iron
Hematoxylin Solution:
Stock Solution A:
Hematoxylin ----------------------------- 1 g
95% Alcohol
----------------------------- 100 ml
Stock Solution B:
29% Ferric chloride in water --------- 4 ml
Distilled water ------------------------ 95 ml
Hydrochloric acid, concentrated ---- 1ml
Weigert's Iron Hematoxylin Working Solution:
Mix equal parts of stock solution A and
B. This working solution is stable for 3 months (no good after 4
months)
Biebrich Scarlet-Acid Fuchsin Solution:
Biebrich scarlet, 1% aqueous --------- 90 ml
Acid fuchsin, 1% aqueous --------------10 ml
Acetic acid, glacial --------------------- 1 ml
Phosphomolybdic-Phosphotungstic Acid Solution:
5% Phosphomolybdic acid ------------- 25 ml
5% Phosphotungstic acid -------------- 25 ml
Aniline Blue Solution:
Aniline blue ------------------------------- 2.5 g
Acetic acide, glacial --------------------- 2 ml
Distilled water --------------------------- 100 ml
1% Acetic Acid Solution:
Acetic acid, glacial ----------------------- 1 ml
Distilled water ---------------------------- 99 ml
Procedure:
1.
Deparaffinize and rehydrate through 100% alcohol, 95%
alcohol 70% alcohol.
2.
Wash in distilled water.
3.
Stain in Weigert's iron
hematoxylin working solution for 10 minutes.
4.
Rinse
in running warm tap water for 10 minutes.
5.
Wash in distilled water.
6.
Stain in Biebrich
scarlet-acid fuchsin solution for 10-15 minutes. Solution can be saved
for future use.
7.
Wash
in distilled water.
8. Differentiate in phosphomolybdic-phosphotungstic acid solution for 10-15 minutes or until collagen is not red.
9. Transfer sections directly (without rinse) to aniline
blue solution and stain for 5-10 minutes. Rinse briefly in distilled
water and differentiate in 1% acetic acid solution for 2-5 minutes.
10.
Wash in distilled water.
11.
Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl
alcohol (these step will wipe off
Biebrich
scarlet-acid fuchsin staining)
and clear in xylene.
12.
Mount with resinous mounting medium.
Results:
Collagen ---------------------------------------- blue
Nuclei ------------------------------------------- black
Muscle, cytoplasm, keratin ------------------- red
Positive Controls:
Skin, lung, stomach, intestine.