[Histonet] Re: Massons Trichrome on decalcified bone

gayle callis gayle.callis <@t> bresnan.net
Wed Apr 4 13:13:26 CDT 2012

Dear Sara,  


You wrote: 

I work at an Orthopaedic research lab and I have been having some trouble
getting our trichromes to work on our bone. I have tried a masson's, gomori,
and goldner's and in all cases the bone stained red.


Our bone is arrive to our lab frozen, we fix them in formalin, embed in
paraffin, and decal with EDTA. I also post fix with Bouins before staining.


Techs that worked in this lab before me were also getting the same red
staining of bone.


Is there something during processing that could cause this reaction? We fix
in formalin, dehydrate through graded alcohols, and clear in xylene.



I doubt your processing has any effect on Mass Tri staining.   However,
incomplete decalcification can but I suspect it may be the staining protocol
itself.   Using a simple weight loss/weight gain decalcification end point
test with EDTA is a good idea, and can be used for acid decalcification
endpoint testing IF you are not presently using an endpoint test.     


Years ago, I visited the AFIP bone lab, and acquired a Massons Trichrome
protocol which worked much better than the standard Massons Trichrome found
in most textbooks.  It never failed to work well with our decalcified bone
work although we did use acid decalcification.    This is NOT a kit method.
All reagents are made up in house, and post mordant heating Bouins is NOT
done in a microwave.  We preferred to let the sections sit in Boiuns
overnight  at RT, or heat in a water bath.   We never used a dry heat,
incubator type oven where one gets uneven heating in the chamber.  


There is more to doing Massons Trichrome on decalcified bone other than just
following the recipe from a textbook. One of the best discussions on
understanding the chemistry/theory of trichrome staining is found in Sheehan
and Hrapchak, Theory and Practice of Histotechnology.    


The AFIP method has some different staining times, plus how to remove the
biebrich scarlet/acid fuchsin from collagen BEFORE going into aniline blue,
a much more controlled staining method.    It also has a modified Weigerts
Iron hematoxylin that is superior, a bit more concentrated since Iron
hematoxylin tends to be removed by the acidic staining solutions.   I will
be happy to send these methods to you privately.  


Gayle M. Callis 




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