[Histonet] Help with Masson's Trichrome - not working on cryo sections

[Jennifer Fricton]

Hello,

I am completely stumped by a problem we¹ve been having with our Masson¹s
Trichrome.  It isn¹t working properly on cryo sections.  We developed our
protocol several years ago and it has been a standard in our lab without any
trouble, until about four months ago when it just stopped working on frozen
sections.  It still works fine on paraffin sections.

You can see images at this link:
http://www.med.umn.edu/lhi/prod/groups/med/@pub/@med/@dom/@lhi/documents/con
tent/med_content_340494.pdf

What seems to be happening is that the beibrich scarlet + acid fuchsin
reagent is either not staining the muscle tissue, or is washing out of the
tissue.  The aniline blue is still staining the connective tissue very well
(it stopped working, too, but we solved by reducing time in water rinse
following stain).  My Masson¹s protocol is very standard ­ essentially
straight out of the Armed Forces Institute of Pathology Manual (p. 94).  I
fix my cryosections in 10% NBF x 30 mins. before starting the protocol.
I¹ve made sure my formalin is fresh and properly stored, with no white
precipitate.  I¹ve tried the Bouin¹s mordant step at both 56C and room temp
overnight.  I¹ve reduced my distilled water rinses following the biebrich
scarlet and aniline blue steps to just a quick dip, thinking maybe I was
de-staining too much before setting the stains with acetic acid.  I¹ve
re-made all solutions, checked all components and pH levels.  I haven¹t done
anything different with my frozen tissues, and I¹ve tried several different
freshly cut tissues.

Has anyone seen this before and have any tips on what is going wrong?  Your
help is greatly appreciated!

-- 
Jennifer L. Fricton, B.S.
Scientist, Lab Manager
Lillehei Heart Institute Histology & Microscopy Core Facility
University of Minnesota School of Medicine, Division of Cardiology
312 Church Street SE
4-290 Nils Hasselmo Hall
Minneapolis, MN  55455
Lab: 612-626-3090