RE: MMA embedding issues
|From:||"Garcia, Vicki [Non-Employee/0111]" |
I have some suggestions for you and can also send you a protocol I have used
with success. I do not have it with me at this time but I will write down
your email address and send it to you tomorrow.
From my experience, the amount of benzoyl peroxide is critical. Too much
and you get bubbles, too little and the solution doesn't set up. Also, I
always incubated mine in a 37 degree water bath overnight. Hope this helps
and I'll send you my protocol tomorrow.
South San Francisco, Ca.
Date: 22 Apr 2003 13:19:32 -0600
From: Anthony Norman
Subject: MMA embedding issues
I seem to have a fairly unique problem, judging by the lack of previous
posts on such a topic.
We have recently managed to spin up an embedding/sectioning protocol
using an MMA/GMA mixture borrowed from a colleague's lab and have had
reasonably good success in the early going, especially once we figured
out how to best dissipate the heat from our larger specimens. However,
recent batches of the embedding media are reluctant to polymerize, or
if they do start, form more a Jell-O block than anything useful. We
haven't been able to isolate the issue to a change in media components,
specifically, and I was wondering if I could pick everyone's brains to
see if we are missing something?
The MMA we are using is from Aldrich and is inhibited with a vague
concentration (10-100ppm) of MeHQ. Our original recipe called for
Fisher's MMA, which was inhibited with 25ppm HQ, but they no longer
carry it apparently. We dissolve PEG-distearate into the MMA prior to
mixing with GMA (10%) and dibutyl phthalate (5%). Wetted (25%) benzoyl
peroxide is added at 8mg/ml to overcome the inhibitor and then use
20-30 ul/ml JB-4 solution B in the final block embedding mix. We use
some small, sealable Rubbermaid containers as molds for the bigger
pieces and 50ml PP conical vials for the smaller pieces. Everything is
placed under vacuum for about an hour, then flushed with nitrogen,
sealed and placed into a chilled water bath to setup.
Thinking the inhibitor might be the issue, we've taken some of the
eariler pre-mix (without BP) and varied the BP concentration using the
worst case inhibitor concentration as the upper bound. That seemed to
help somewhat, though we still get mixed results and a substantial
gooey layer in most of them. The original recipe seems to remain a
solution for days in the waterbath and, if warmed to room temp, will
eventually polymerize in an impressive display of bubbling. Sometimes,
it never sets up.
Since this began to be an issue for the smaller specimens we were
embedding, we tried increasing the JB-4 addition toward the upper
limit, again with less than ideal results. In general, the lower
portions of the blocks tend to polymerize while a significant amount of
material remains at the top. On the up side, we don't have any bubbles
in the blocks.
The final bit to the puzzle is that the color of the blocks have
gradually moved from a clear amber to clear colorless, though I am not
as concerned about this part, since when they are solid, they seem to
section as well as the earlier blocks.
We are planning to try the new MMA offering through Fisher, by ACROS, I
believe and possibly that sold by Fluka, so as to nail down the
different inhibitors used. Most of the components are new, ordered
through EMS or Sigma-Aldrich.
Any thoughts on what else might be the problem? Can the MeHQ be washed
from the MMA in the same fashion as the regular HQ? We don't have a
separatory funnel at present, but could probably scrounge one up
somewhere. It's getting a little old just mixing solutions and
watching them set up as we try to isolate the troublemaker and my
technician has better things to be doing with her time.
Thanks in advance,
UW- Orthopaedics and Sports Medicine/Bioengineering
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