[Histonet] Re: Poor Weigerts Hematoxylin staining with Massons Trichrome

Fri Sep 17 11:40:13 CDT 2010

In general, we found the original/classic Weigerts iron hematoxylin stain to
be weak and almost washed out of tissues after staining with Massons
Trichrome. We no longer use the original formula, but a more concentrated
modified formulation that is not differentiated out as much by the acidifed
solutions found in Mass Tri.  We also found this problem with Massons
Trichrome kit components, where companies probably package the original
method's solutions.  

We make up Weigerts fresh each time, and if it will last for a week for you,
fine......... but our work tends to be a one time staining with Mass Tri,
then weeks before it was done again.  The problem is: ferric chloride
continues to oxidize the hematoxylin throughout over time, that week or
longer, weakening the iron hematoxylin staining capabililty.   This is
discussed in Sheehan and Hrapchak Theory and Practice of Histotechnology.
Acid decalcified bone presents even more of a challenge, since nuclei
(DNA/RNA) in cells are hydrolyzed by acid decalcifiers, compromising
staining of nuclei, a problem seen with routine H&E staining.  Deanna was
correct on her assessment of this stain for best results.  

This modified  Weigerts Hematoxylin was published in J of Histotechnology in
a paper on Kreybergs stain on skin.  The second Extra Strength Weigerts was
found on Histonet years ago and I have not tried the latter.  I suggest you
see which one you prefer, as we use the first Modified Weigerts.  Over the
years, the modified gave us far superior nuclear staining with Massons
Trichrome.  

Weigert's Iron Hematoxyin MODIFIED (found in J of Histotechnology in a
method for Kreybergs stain on mouse skin).  

Solution A. 

            2% Hematoxylin in 95% ethanol

Solution B. 

            62% Ferric Chloride   4 ml

            Distilled water           95 ml

            Hydrochloric acid      1 ml

Mix equal amounts of Solution A and Solution B 

MIX FRESH JUST BEFORE USE AND DISCARD AFTER USE.

Extra Strength Weigerts Iron Hematoxylin from Histonet (reference unknown,
but supposedly from J of Histotechnology and method originated by Mabel
Myli, Mayo Clinic) 

 Solution A:  Hematoxylin       10 g

                    95% ethanol      100 ml

Solution B:             11.6 g Ferric Chloride
                              980 ml Distilled water
                               10 ml 25% hydrochloric acid

Working Stain Solution
               Solution A                             5 ml
               Solution B                             25 ml
               Absolute Ethanol                  20 ml

Staining time for both of these formulations is 10 minutes, followed by
rinsing for 10 min in running tap water (hematoxylin will be black)

Gayle M. Callis
HTL/HT/MT(ASCP)

************************
You Wrote: 

I do Weigert's staining for 10 minutes and use it for a week at the most.
I, 
too, have heard conflicting times about the stability of the Weigert's, but
have 
the best results with using it no longer than a week.

Deanna Rhoads HT (ASCP)

________________________________
From: Andrea Marion <amario3
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t> uic.edu>
To: histonet  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
<@t> lists.utsouthwestern.edu
Cc: itai.moshe  <http://lists.utsouthwestern.edu/mailman/listinfo/histonet>
<@t> mail.huji.ac.il
Sent: Fri, September 17, 2010 10:31:16 AM
Subject: [Histonet] Re: Masson's trichrom - problem with nuclei staining

Hi Itai,

I am interested to hear if you've resolved this problem.  We use the same
kit to stain mouse heart and embryo sections, PFA fixed. Our protocol is
similar to the one you mentioned. I cannot get nuclei staining with this
method either. The nuclei are well stained (ie black) up to the PMA/PTA
step, but during that step the nuclear stain is completely removed. I
cannot shorten the PMA/PTA step without negatively effecting the collagen
stain.In general, we found the original/
I have in our original protocol that the Weigert's working solution is
good for one month, but I cannot recall if this is from Sigma's
specification sheet or a personal observation from a lab member.  However,
from reading online some say it is good up to 4 months, others say it
needs to be prepared fresh each time. I have tried fresh preparations with
the same results.

My instinct is that something is off - the staining is just not stable
enough to withstand the subsequent acid steps in Masson's trichrome. Can
an expert weigh in on this? Is there a way to strengthen nuclear staining
from Weigert's?

Sigma's formulas and usage recommendations are:

Part A: 1% w/v certified Hematoxylin in ethanol
Part B: 1.2% w/v Ferric chloride, 1% v/v Hydrochloric Acid

Combine equal volumes Part A and Part B, stain sections for 5 minutes.

Andrea Marion
Graduate Student
University of Illinois at Chicago
amario3 /at/ uic /dot/ edu

Itai Moshe itai.moshe <@t> mail.huji.ac.il
Wed Sep 15 11:34:51 CDT 2010

Hi Histonet's

I'm using Masson's trichrome to stain paraffin mice diaphragm fixed with
PFA.
I'm using this protocol:
http://www.bcm.edu/mcb/rosenlab/index.cfm?pmid=12997
<http://www.bcm.edu/mcb/rosenlab/index.cfm?pmid=12997>With sigma masson's
kit (#HT15) and Weigert's Iron Hematoxylin Set (#HT1079)
The staining is beautiful, but i can't see the nuclei good enough.
1) Is there a way to enhance the nuclei staining ? (the nuclei is the only
reason that i"m not using the simpler sirius red and fast green staining.)
2) What is the meaning for washing in running tap water washing, is it done
by putting the slides in a  jar with simple tap water for a few minutes ?

Thank's
Itai