[Histonet] Staining of plastic embedded samples

Gayle Callis gcallis <@t> montana.edu
Mon Oct 24 10:52:31 CDT 2005

I presume these are undecalcified bone samples, as Technovit 9100 is the 
methylmethacrylate version.  After the final polishing of your thicker 
sections, etch the surface of your polished block, using an ultrasonicator, 
with 1% formic acid for 30 sec to 1 minute.  Rinse well with running tap 
water, dry the section and immerse into your stains.  You may have to 
adjust the times, but MSB may not be the ideal stain for ground, polisher 
MMA embedded and then acid etched sections.  It may be a better stain for 
microtomed, thin MMA sections.

You can also try etching the surface with 95 to 100 % ethanol, to soften 
the plastic and allow your stains to penetrate into the soft tissues but 
you need to do this at the beginning of staining protocol or you may remove 
other dyes you have applied to the section.   Basically, with the acid 
etch, you are doing a mild surface decalcification for only a few 
micrometers to allow the stains to penetrate into the bone, with alcohol 
etch,  you are only softening and NOT removing the plastic to permit these 
small molecular weight dyes to penetrate into the soft tissue.  Whatever 
you do, do not over etch with acid longer than 1 min and do not use a 
strong mineral acid such as hydrochloric acid to etch, you will remove too 
much calcium at a greater depth (more micrometers) and you can have some 
overstained sections.   If you make a mistake, you can always polish off 
the stain, but do not grind too much or you may remove the area you want to 

Just be aware that if you acid etch, your staining times may change with MSB.

A toluidine blue stain developed for bone surface staining by Eurell and 
Sterchi is excellent for this purpose, as is MacNeals tetrachrome, but on 
the latter use an inhouse prepared stain solution, commerical stain doesn't 
work well at all.

At 06:15 AM 10/24/2005, you wrote:
>I´ve got a problem with staining plastic embedded samples.
>The samples are embedded in Technovit 9100 new. The blocks are polished 
>and sticked on a plastic
>slide. With a hand saw I cut them and the slides are getting polished 
>again and afterwards they
>are getting stained.
>Now my problem: I do the Masson Goldner or MSB stain, but the lightgreen 
>or methyl blue doesn´t
>appear in the colored slice. Both are only red-brown.
>I don´t remove the polymer (with 2-methoxyethylacetat) before staining, 
>because of the plastic
>slides. They also dissolve.
>I hope anybody could help me.
>Thanks Nicole
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu

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

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