[Histonet] Masson's Trichrome Troubleshooting

[Gudrun Lang]

Hi,
Check the staining after the PTA/PMA step. The acid should destain all the
tissue areas, where afterwards the anilinblue should bind. The acid replaces
the Biebrich scarlett in this areas and enables and enhances the binding of
anilinblue.
If the differentiation in insufficient, it may be the quality of the acid to
be the culprit. The pH should be about 2, if I remember correctly.

Check the temperature of the bouin after the microwave. If you cook the
tissue, it may also change the stainability. Microwave is not microwave, the
instrument in your old lab may have different characteristics.
A softer way is 60° for two hours in the oven.

Gudrun

-----Ursprüngliche Nachricht-----
Von: Campbell, Tasha M. via Histonet
[mailto:histonet at lists.utsouthwestern.edu] 
Gesendet: Mittwoch, 30. Mai 2018 14:15
An: histonet at lists.utsouthwestern.edu
Betreff: [Histonet] Masson's Trichrome Troubleshooting

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

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