[Histonet] Troubleshooting Masson Trichrome Stain

[Anna Coffey]

Hello Histonetters,

We regularly perform the Masson Trichrome stain in our lab and recently
have been noticing some inconsistent results. The main issue seems to be
that the Biebrich Scarlet is not being adequately removed by the PTA/PMA,
as many of the areas containing collagen are showing up as purple instead
of bright blue. We use fresh reagents every time we run the stain and our
protocols are as follows:

Rehydrate (xylene --> 100%EtOH --> 95% --> 80% --> 70%)
Bouin's in microwave (1 min)
Stand at RT in Bouin's (15 min)
Running tap (5 min)
Weigert's Hx (10 min)
Running Tap (5 min)
Biebrich Scarlet (1.25 min)
Rinse dH2O
Phosphotungstic acid/Phosphomolybdic acid (10 min)
Aniline Blue (10 min)
Rinse dH2O
1% Acetic acid (1 min)
Rinse dH2O
Air dry for dehydration

Has anyone else experienced this same problem and, if so, what corrective
actions have worked for you? I'd greatly appreciate your advice!

Thanks,
Anna
-- 
Anna Coffey
Senior Histology Technician
Department of Oncology
Histopathology and Tissue Shared Resource
LR-10 Pre-Clinical Sciences Building
Lombardi Comprehensive Cancer Center
Georgetown University
202-687-7890
ahc53 <@t> georgetown.edu