Collagen Staining Protocol

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Guy Sovak
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Collagen Staining Protocol

Massons Trichrome staining Collagen Fibers

This methos is a bit complicated but gains good and detailed results,
Collagen fibers are visualised in blue.

One can uused ether Parafin Section (7-10uM) or Frozen sections.

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. The
working solution is stable for up to 3 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

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 7-10 minutes.
4. Rinse in running warm tap water for 10 minutes (in couplin jar).
and Wash twice in distilled water.
5. Stain in Biebrich scarlet-acid fuchsin solution for 15 minutes.
6. Wash twice in distilled water.
7. Differentiate in phosphomolybdic-phosphotungstic acid solution for 15 minutes or until collagen is not red.
8. Transfer sections directly (without rinse) to aniline blue solution and stain for 10 minutes. Rinse quickly in distilled water and differentiate in 1% acetic acid solution for 2-5 minutes, Wash in distilled water.
9. Dehydrate very quickly through 95% ethyl alcohol, absolute ethyl alcohol (these step will wipe off Biebrich scarlet-acid fuchsin staining) and clear in xylene, ( option to use methyl green counter stain).
10. Mount mounting medium ( dont use DAPI containing medium).

Collagen will be stained blue
Nuclei stained black
Muscle, cytoplasm, keratin stained red