Re: [Histonet] Problem with Masson's Trichrome

From:Gayle Callis

One thing, I do not think you have a correct Massons Trichrome staining 
procedure as Bouins is absolutely crucial to success of Massons Trichrome - 
Weigerts Iron hematoxylin is the hematoxylin of choice (black nuclei).  In 
fact, most histotechnology books say Bouins is fixative of choice for Mass 
Tri, or if your tissues are fixed with PFA you are using, you MUST postfix 
with this fixative prior to staining at either 56C for 1 hour or let 
sections sit in this overnight at RT (this is nice as you do not have 
heated Bouins fumes).  You can continue to use your fixative, but must do 
the Bouins step.  You can buy Bouins ready to use from Sigma and other 
supplies - advisable as Bouins  contains picric acid, saves you time and 
having to store a hazardous substance.  The Weigerts iron hematoxylin is 
best when made up fresh each time.  It continues to oxidize, and if kept a 
long time as a working solution, loses strength, resulting in weak nuclei 

You should access a histotechnology textbook and read the theory of Masson 
Trichrome stain (your university library).   The reason for Bouins and 
acidified staining solutions (aniline blue) is the acid pH is necessary to 
increase the selectivity for the collagen fibers.   It is hard to explain, 
maybe John Kiernan or someone else will expound on this.   Hrapchak and 
Sheehan Theory and Practice of Histotechnology has a good discussion on 
mechanism of Mass Trichrome staining.

The stain is straight forward and easy to do, uterine tissue stained with 
Mass Tri is beautiful -  a good example of how the stain works - blue 
collagen and mucin,  red muscle fibers, cytokeratin and cytoplasm, and 
black nuclei.   If I remember correctly, many, many years ago, Mass tri 
stained uterus was required for ASCP HTL exam.

Your brittle tissue may be do to over exposure to alcohols, clearants, or 
heat of paraffin and not because of stain protocol.  If the tissue is not 
totally fixed prior to processing, alcohols will complete fixation and dry 
the tissue excessively.  Check fixation time and your processing schedule - 
you did not indicate which species your were working with?

At 01:19 AM 8/6/2004, you wrote:
>Hi Everyone!
>I was recommended to try the Masson's Trichrome stainings on the slides of 
>uterine tissue that I have been collecting (Previously I had only 
>performed Hematoxylin & Eosin stains). This is a new method to our lab and 
>thus far, staining has not gone well. Although my sections contain a 
>significant amount of muscle, I have only been able to see the aniline 
>blue stain. On a couple of the slides, there was a slight dark purple 
>coloration on the epithelial lining.
>I have noticed upon comparing the protocol we received to others found on 
>the internet that ours lacks the step in which  the slides are placed in 
>Bouin's fluid. There was also no specification in our protocol for the 
>type of hematoxylin, so we had been using the same hematoxylin (Harris's 
>modified with acetic acid) that we use in our H & E stainings. I was 
>wondering if any of these might have led to the stainings not going as 
>planned. The tissue sections also looked slightly brittle, perhaps due to 
>a harsher stain. I am wondering if this has anything to do with my 
>fixation process in which I use a 4% paraformaldehyde/PBS mixture, ethanol 
>and xylene steps, as well as embedding the tissues in paraffin.
>Is Bouin's a crucial step, would I have better staining with a different 
>tissue fixation, or should I be looking in a different direction? Any 
>suggestions would be very much appreciated!
>Amber Greenbank
>Arizona State University
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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