FW: [Histonet] Troubleshooting Masson Trichrome Stain

[Elizabeth Chlipala]

Sorry forgot to reply all.

Anna

Do you make up your solutions fresh especially the PTA/PMA and aniline blue.  We don't run the microwave method we heat in an oven in bouins for 1 hour, but we make sure that the bouins is at 60C prior to us placing the slides in the solution.  We stain longer in the Biebrich scarlet  (20 minutes) we only use the PTA/PMA once and then about 2 minutes in the Aniline blue.  We found that it's important to use fresh reagents, we get the best consistency of staining with all over fresh reagents.  Since you are moving from the PTA/PMA right into the aniline blue, if you use the aniline blue over and over again the pH will change because you are carrying some PTA/PMA over on the slides, this will cause problems with the stain.  We are not like a clinical lab so we could staining up to 100 or so trichrome slides at a time so making sure we have fresh reagents is key for consistency for our samples.  Good Luck, one other thing the trichrome stain is one of the more difficult histology stains to run correctly.  A good way to tell if the stain has worked properly is to look at smaller vessels.  The smooth muscle of the smaller vessels should be red, and the connective tissue blue, frequently you will see the smooth muscle of the vessels stain a bit grey that's when you know that that stain has not worked.  The stain needs to be balanced with neither component overpowering the other.

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308
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liz <@t> premierlab.com
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-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Anna Coffey
Sent: Tuesday, February 11, 2014 12:44 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Troubleshooting Masson Trichrome Stain

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
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