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here is a trichrome procedure i use for plastic sections, uses no phos.

Submitted by Patsy Ruegg, HT(ASCP)QIHC    UCHSC Retired

Dry sections on 70dc hot plate for at least 20 minutes or 30 min
(62dc oven)

Mordant slides in saturated picric acid

Wash in running tap water                                             10

Rinse in deionized water

Stain in extra strength Weigert's  - filtered                       5 min

Rinse in running tap water                                           3-5

Stain in beibrich scarlet - acid fuchsin                           30 min

Rinse quickly in deionized water

Stain in 2% lt. green in 2% acetic acid                               10

Rinse in deionized water

3% acetic water for 4 to 5 min will enhance the blue

3% acetic in 95% alcohol, 2 changes, to remove stain from plastic media

Two changes of acetone

Clear in xylene and coverslip in permount

Results:  Nuclei-black; mineralized bone-green; osteoid-red/orange;
collagen-green or blue (depending on counterstain); keratin, muscle, and
intracellular fibers - red

Biebrich scarlet-acid fuchsin
  2% biebrich scarlet
  1% acid fuchsin
    Mix together:
      45 ml  bieb-scarlet  (Can be reused but filter before each use)
       5 ml  acid fuchsin
      Stir, then add 0.5 ml acetic acid

Extra Strength Weigert's
  Sol A  10 gm  Hematoxylin
            100 ml  95% alcohol
  Sol B   11.6 gm ferric chloride
            980 ml deionized or distilled water
              10 ml  25% HCL acid
Mix together and filter:
  Sol A    5 ML
  Sol B   25 ML
  Abs. alcohol  20 ml

Aniline Blue
  2.5 gm aniline blue
  2 ml acetic acid
  100 ml deionized or distilled water

2% Light Green
  2 gm light green
  98 ml distilled water
  2 ml acetic acid

(Procedure orginally developed by Mabel Myli, Mayo Clinic many moons

Phi-Huynh Su wrote:

> Dear Histonet,
> 1.  I have problems staining tendon with Masson Trichrome.  It seems
> like parts of the fibers were dissolved by the method.  I've stained
> same set of tendons with Alcian Blue, pH 2.5, and there was no obvious
> change.  So I'd assume that acetic acid wasn't at fault.  I'd think
> the phosphomolybdic acid was the culprit.  I have repeated the
> procedure several times.  When I stained femoral head sections with
> MT, it worked nicely.  Please help.  Is there any substitute for
> phosphomolybdic acid? Tissues were fixed in Bouin's solution.  Since
> the tendons are quite thick, 5-8mm, they were fixed for 3 days.  The
> excess picric acid was removed after rehydration of the tissues prior
> to hematoxylin step, not prior to processing.
> 2.  What will happen if I forgot to shake the tissues for the first 12
> hours of fixation?  (Bouin's fixative solution was used.)
> Thank you in advance

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