Re: [Histonet] staining MMA

From:Gayle Callis


What is Richardson's stain? What dyes does it contain?

It more than likely is the failure of a dye's  (probably of high molecular 
weight) being able to penetrate MMA plastic in order to stain 
collagen.  There are some ways to help this along, etching with solvents to 
soften plastic is one way.  If it is bone in PMMA, one can 1% formic acid 
etch for 1 min, rinse well and stain the surface.   With thin sections, 
total removal of plastic probably solves the problem.

You should read this publication - Staining of plastic sections: a review 
of problems, explanations and possible solutions.  RW Horobin, J Microscopy 
131(2):173-186, 1983.   This article will help you understand the problems 
you encounter and how to try and solve them.  PMMA is very hydrophobic, GMA 
is also hydrophobic but less so than MMA.   Dyes with low molecular weight 
penetrate MMA better than others, these are basic fuchsin, light green, 
toluidine blue, Stevenels blue or Sandersons rapid bone stain  (potassium 
permangante oxidized methylene blue produces T blue, thionin, azures, etc), 
the dyes in McNeals tetrachrome,  and methylene blue/basic fuchsin 
comination.    High molecular weight dyes probably will require MMA 
removal, really only possible in a thin section mounted on a slide unless 
you try solvent etching.

Did you remove the MMA from your 5 um thick sections?  If so, then dyes can 
get to the fibers since the MMA is gone.  Horobin discusses collagen fibers 
and how one can reverse colors in trichromes when staining MMA embedded 
tissues - very interesting chemistry.

There are other factors involved, heat, pH, thickness of section.

Have you ever tried doing a surface stain of your 50 um sections using 
McNeals Tetrachrome (do NOT buy this as a commercial solution, make it in 
house) - use it alone or combine it with toluidine blue as a double 
stain.  Brilliant results.

   () At 09:00 AM 1/19/2005, you wrote:
>Hello everyone
>this is my question about a titane implant embeded with MMA (like always 
>!!!) :
>I done a thik section  with titane and polished to 50um stained with 
>richardson's stain and i see a tissue with no fibers of collagen directed 
>towards titane.
>and when i remove the titane, and then i can do sections of 5um thikness, 
>staining with goldner trichrome, i see collagen fibers directed towards titane.
>do you think if it's because of the stain not appropriated with fiber 
>collagen with thik section about 50 um ?
>Or something else ?  is it possible to stain very thin collagen fibers in 
>sections about 50 um thinkness.
>Thank you very much for any helm and advice.

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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