Re: Question about biomaterial immunohistochemistry


Are you using a Tungsten Carbide knife to cut with?  I prefer D profile TC
knives to C profile TC knives.  I have some experience cutting hydroxyapatite
and found it to be very difficult because it is so brittle.  I use the enzyme
histochemical methods for acid and alkaline phosphatase and have no loss of
enzyme with GMA embedding using benzoyl peroxide as the activator.  The alkaline
phos enzyme is much more sensitive to heat than acid phos in my experience, so
be careful about controling heat during polymerization and even heating the
sections after they are cut.  If you can't get knife cut sections you might have
to go to ground sections or diamond wire saw cut sections.
Patsy Ruegg

Tithi Dutta Roy wrote:

> Hi everyone,
> I have some questions about performing an immunohistochemical stain on
> PMMA-embedded rabbit skull bone containing a hydroxyapatite and/or composite
> biomaterial.  One of my concerns is whether the blade that we have to cut
> samples will cut through the biomaterial.  Has anyone ever had problems
> using a Reichert-Jung Polycut E microtome to cut PMMA?  Also, I would like
> to stain for either alkaline or acid phosphatase.  The embedding process was
> done at room temperature until the end when it was polymerized at 37 degrees
> C in a vacuum oven.  My practice samples are all embedded in PMMA with 2.5%
> benzoyl peroxide.  Will the enzyme activity have been destroyed?  I am also
> concerned if the tissue within the biomaterial will stain, if the
> biomaterial is dense like hydroxyapatite.  I would like to not have to
> decalcify the sample since the biomaterial remaining in the sample is pretty
> important to me.  If ANYONE has any ideas at all, please let me know.  I am
> working blind here, I have done lots of reading but I wanted to know if
> anyone else had any ideas before I started.  Thanks!
> Tithi
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