[Histonet] Trichrome troubleshooting

Gayle Callis gayle.callis at bresnan.net
Tue Jun 30 16:05:39 CDT 2015

Years ago, I was taught by Jerry Fredenburg, a stain guru, never to
microwave the Bouins step but do this for 1 hour at 60C or overnight at RT.
The post-fixation/mordant is very important in order to acidify the
connective tissue fibers properly for a trichrome stain, and the short time
in MW will not do the job.   Liz is correct here in letting the sections
stand longer in Bouins after microwaving or simply just do the 1 hour at

I have removed Gills type hematoxylins from nuclei  by over exposure to
acetic acids so remember that ALL Masson's Trichrome reagents are acidified
with acetic acid and will automatically do this, even on Weigert's Iron
hematoxylin.     We also examined our sections microscopically during
staining to make sure the check the depth of red staining reagents on smooth
muscle is correct - once again, Liz is correct about this fact.   

Gayle Callis   

-----Original Message-----
From: Elizabeth Chlipala [mailto:liz at premierlab.com] 
Sent: Tuesday, June 30, 2015 2:30 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
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
liz at premierlab.com

March 10, 2014 is Histotechnology Professionals Day

Ship to Address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504

-----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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