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

[Tony Henwood]

Jennifer,

Looking at the images, is it possible that the slides were air-dried for too long prior to staining?

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jennifer Fricton
Sent: Wednesday, 1 June 2011 2:28 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Help with Masson's Trichrome - not working on cryo sections

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

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