[Histonet] Staining of plastic embedded samples
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.
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