[Histonet] Masson's Trichrome Troubleshooting

[Campbell, Tasha M.]

Hello all,

I am having issues with my trichrome stain and I am about to lose my mind!  We just started doing it in house (although at my previous job I had done trichrome by hand for years so I am not a stranger to it. And I never had issues with it).  When I brought it in house at my current lab, I ordered the same kit that I was familiar with.  Its PolySci.  I did the stain about 5 or 6 times and then all the sudden it quit working.  There was red where it should be blue.  And there was blue staining but it was all in the crypts.   I tried tweaking the stain a few times and nothing worked so I got a new kit.  The first 2 times I used the new kit, it worked perfect!  But after that it is back to doing the same thing again!  The collagen is not staining blue.  It is staining red.  Can anyone please tell me why this is happening?  I never had this issue before!  Thanks in advance!! See my protocol below.

1. Mordant in Bouin's solution, microwave 5 minutes, allow to stand 15 minutes.
 2. Wash in running tap water to remove the picric acid, 5 minutes.
 3. Weigert's Working Hematoxylin, 10 minutes.
 4. Blue in running tap water for 5 minutes, rinse in distilled water.
 5. Biebrich scarlet for 5 minutes.
 6. Rinse in distilled water.
 7. Phosphotungstic/phosphomolybdic acid for 10 minutes.
 8. Transfer directly into Aniline blue for 5 minutes.
 9. Rinse in distilled water.
10. 1% Acetic acid for 1 minute.
11. Quick rinse and air dry.
12. Coverslip




Tasha Campbell, B.S.,HTL(ASCP)
Frederick Gastroenterology Associates
310 W. 9th St.
Frederick, MD 21701
301-695-6800 ext. 144