Masson's Trichrome


Results: Nuclei - blue-purple
             Cytoplasm, keratin, muscle fibers - red
             Collagen - blue

Solutions List

Bouin's Solution:
    30 ml Picric Acid
    10 ml Formaldehyde, 37-40%
      2 ml Glacial Acetic Acid

Celestine blue hemalum nuclear stain:

Solution A:   Ferric ammonim sulfate (iron alum)
                    1 g Celestine blue B
                    100 mL DI water
                    7 ml Glycerin

Dissolve the ferric ammonium sulfate in the DI.
Add Celestine blue B and boil for 5 min.
Cool, filter and add Glycerin
Store in brown bottle. Sol'n should be stable for several months

Solution B:   Mayer's Hematoxylin

Biebrich Scharlet-Acid Fuchsin Solution
    90 ml 1% Biebrich scarlet, aqueous
    10 ml 1% Acid fuchsin, aqueous
      1 ml Glacial Acetic Acid

5% Phosphotungtic Acid in DI water

Aniline Blue Solution
    2.5 g Aniline blue
    100 ml DI water
        2 ml Glacial Acetic Acid

1% Glacial Acetic Acid in DI water


Staining Procedure:

  1. Preheat Bouin's solution in glass Coplin jar, in a 56 C waterbath for at least 15 min prior to mordanting,
    step 4.
  2. Deparaffinize and hydrate sections to DI water.
  3. Mordant in preheated Bouin's solution for 1 hr. at 56 C.
  4. Cool for 5 min. and Rinse in running DI water until yellow color disappears, (about 5 min.)
  5. Place sections in Celestine blue hemalum solution A for 10 min.
  6. Rinse with tap water
  7. Place sections in Celestine Blue hemalum solution B ( Mayer's hematoxylin ) for 10 min.
  8. Wash in running tap water for 3 min. Rinse in DI.
  9. Place in Biebrich scarlet -acid fuchsin for 1.5 min. (save sol'n). Rinse in DI
  10. Place sections in 5% Phosphotungtic Acid for 15 min. (discard sol'n)
  11. Place slides directly into Aniline Blue solution for 3 min. (save sol'n). Rinse in DI.
  12. Place in 1% Glacial Acetic acid for 4 min.
  13. Dehydrate quickly through 2 X 95% alcohol, 2 X of abs. alcohol, 6-8 dips each. Clear 3 X xylene and Mount.