[Histonet] Trichrome troubleshooting

Bernice Frederick b-frederick at northwestern.edu
Wed Jul 1 07:28:06 CDT 2015

Actually, I have microwaved the  Bouins (and still do) for small numbers of slides and the results are the same. I ran a liver bx both ways as well as larger tissue to compare. I use the 10 slide plastic coplin jar and generally have 5 or less slides when I do this. One microwaves the Bouins for 30 seconds on power level 7 in a lab grade microwave. A higher level will cause the Bouin's to spill. Slides are then added and left for 5 minutes.  Excess rinsed out and then proceed as per your SOP.  As for the blue- I rinse out excess and do 1 dip in 1% GAA. Rinse and dehydrate (10 dips each solution)

Bernice Frederick HTL (ASCP)
Senior Research Tech
Pathology Core Facility
Robert. H. Lurie Cancer Center
Northwestern University
710 N Fairbanks Court
Olson 8-421
Chicago,IL 60611
b-frederick at northwestern.edu

-----Original Message-----
From: Elizabeth Chlipala [mailto:liz at premierlab.com] 
Sent: Tuesday, June 30, 2015 3:30 PM
To: Suzanne Martin; histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] Trichrome troubleshooting


How many times have you used the kit and reagents, I did look up how the kit works but the trichrome stain can be tricky.  First of all you need to make sure that the mordant (bouins solution) is at 60C prior to placing your slides in them.  We normally heat up our bouins for at least an hour prior to placing the slides in the solution.  We leave in bouins for an hour and a half rather than an hour.   I see that this is a microwave protocol I cannot comment on that but I don't think that the hematoxylin is the issue, if you leave longer in 1% acetic acid that may pull some of the blue stain out or I would try dehydrating with lower alcohol percentage that can pull some of the blue stain out.   I would also try leaving it a bit longer in the bouins after you microwave it - that might help.

Trichrome staining works best with fresh reagents so if you have used these reagents too much that could cause problems.  I'm also not a big fan of the one step trichromes, they are quicker but sometimes not as good as the two steps, just my opinion.

FYI - to evaluate your staining look for a smaller vessel, the smooth muscle should be nice a red, if its greyish or blue you have not done the stain properly.  Good Luck


Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308
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liz at premierlab.com

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-----Original Message-----
From: Suzanne Martin [mailto:smartin at lcpath.com]
Sent: Tuesday, June 30, 2015 12:37 PM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] Trichrome troubleshooting

Hi all,

We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange. 

We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts. 


Thank you.

Suzanne HT

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