[Histonet] Masson's Trichrome Troubleshooting

Thu May 31 10:14:12 CDT 2018

Tasha,

Are you using the PolySci kit k037?  Did they change the formulation of the Bouins?  They do make one that has no picric acid, and doesn't last long.  If so, add a some saturated picric acid to the Bouins to see if that helps.
Are you using the Bouins again after microwaving?  If so, that may be your problem.  
Don't re-use the Phosphotungstic/phosphomolybdic acid, more than 2x.  It sets and diff's the red.

After the Anile blue, is the tissue blue?  If not, then it's the ppa/pma is the problem.  
Use the acetic acid more sparingly.  That diff's the blue some and can take more of it out.  I do it by sight.  Carson's says 3-5 min, but that is way too long.
We don't air dry, but that wouldn't take out the blue.  
Let me know if this helps.

Toysha N. Mayer D.H.Sc., MBA, HT(ASCP)
Instructor/Education Coordinator
HTL Program
MD Anderson School of Health Professions
713.563.3481
tnmayer at mdanderson.org

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Message: 2
Date: Wed, 30 May 2018 12:15:22 +0000
From: "Campbell, Tasha M." <tmcampbe at fmh.org>
To: "histonet at lists.utsouthwestern.edu"
	<histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Masson's Trichrome Troubleshooting
Message-ID:
	<C0FE6645D0D08D46A71C8F545654B38E5419A743 at EXCH1.fmhnt.fmh.org>
Content-Type: text/plain; charset="us-ascii"

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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Message: 5
Date: Wed, 30 May 2018 15:47:39 +0200
From: "Dr. Michael Gudo (Morphisto GmbH)" <michael.gudo at morphisto.de>
To: "Campbell, Tasha M." <tmcampbe at fmh.org>
Cc: "histonet at lists.utsouthwestern.edu"
	<histonet at lists.utsouthwestern.edu>
Subject: Re: [Histonet] Masson's Trichrome Troubleshooting
Message-ID: <5661355A-5F32-46EB-9F53-79FC9E49477D at morphisto.de>
Content-Type: text/plain;	charset=utf-8

Dear Tasha,

well, I think you should modify your protocol:

(1) don?t use microwave
(2) wash in ethanol after Bouin and not in water (70 - 80 %)
(3) tap water as long as Weigert working solution
(4) Bieberich Scarlet is not the correct stain for Masson, you should use Acid fuchsine + Ponceau 2R + Azophloxine
(5) use a higher concentration of phosphortungstic acid, or / and least leave the sections much longer, up to 30 minutes in this step
(6) do not ?air dry?, but remove water with ethanol and ethanol with xylene, then you should get very nice results.

Well, and finally, if you allow I would like to offer you the Masson Kit / Masson Goldner Kit our company offers for quite brilliant stainings: 

https://webshop.morphisto.de/catalogsearch/result/?q=Kit+MASSON+trichrom <https://webshop.morphisto.de/catalogsearch/result/?q=Kit+MASSON+trichrom>

Please do not hesitate to contact us, if you have any further questions.

With best regards
Michael

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