[Histonet] Re: bubbles around tissue in MMA

[Gayle Callis]

Lori,

Bubbles often indicate there is rapid polymerization at too high a 
temperature.

If you tissue is properly infiltrated, embedding can be in polypropylene 
vials (your stents are probably small) or some other polypropylene tube 
with a cap on it.  I have even used conical 15 ml centrifuge tubes - you 
can cut the end off, push block out.  Some find vials or tubes that have 
o-ring design within the cap - for a good seal.  All major vendors have 
these - shipping tubes or storage vials.

An open embedding mold permits excessive monomer evaporation, you get 
funky, hard to cut blocks since you have changed the ratio of your MMA 
components.  Use a sealed tube, scintillation vials have been used.  It is 
nice to have a grinder to shape a block prior to cutting and get extra 
plastic trimmed away down to sample to save knives and time.  We tried Peel 
away molds but the blocks were horrible, since they were open at the top 
during polymerization.

You really don''t need to pull a vacuum before or during 
polymerization.  If the tissue is perfectly infiltrated, the MMA around it 
will polymerize along with what is in the tissue.

Also, you can make  a prepolymerized layer of MMA on the bottom of a tube, 
lay the infiltrated tissue on top of that and add your embedding MMA,  the 
nonpolymerized monomer forms an interface with the prepolymerized layer and 
actually aids in polymerization, a start so to specak.  Smooth, clear, 
bubble free  polymerization occurs.   This worked very well for us many 
times.  Make sure the temperature at polymerization IS not too warm.  We 
had bubbles if we increased the temperature to 40C, although waterbath 
polymerization at 37C overnight was trouble free.

We made prepolymerized layers from the last infiltration mixture rather 
than dispose of that.  Just fill tubes,  tightly cap, stack them  and let 
them sit around to polymerize. Nice way to dispose of waste MMA and have 
ready made prepolymerized layers.   It also aids in having a flat faced 
block in the end.

  After embedding let your samples sit at RT in a hood, somehow this "rest" 
seems to allow bubble free polymerization - then go to a 37C waterbath, DO 
NOT use an incubator.  Incubators do not have even heating due to 
prevailing air currents and you can have bubble trouble - big time.

At 06:44 PM 1/26/2005, you wrote:
>Hello all, this is my first query to the histonet. I recently started 
>embedding tissues in MMA using the Technovit 9100 kit. I will mostly be 
>embedding stented vessels. I have had problems with bubbles forming around 
>the tissue during polymerization. I have not tried destabilizing the 
>monomer yet, but did try using a vacuum before placing the sealed samples 
>at 4 degrees C in a dessicator. Any ideas you have would be welcome. I did 
>not find anything on this in the archives. Also, I would like to know what 
>people use for embedding molds. Thanks,
>Lori
>
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Gayle Callis
MT,HT,HTL(ASCP)
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)