[Histonet] MMA

[Nancy Maronto]

Myriam,
I  have used the MMA/butylmethacrylate/methylbenzoate/peg 400/ benzoyl peroxide (BPO) combination to embed undecalcified bone and small stented arteries.  It is an easy combination to make up and use.  I expect you have three changes suggested in your procedure.  Mix each about 4 hours before use.  It seems I have the best results with this.  The infiltration time will have to be worked out and if vacuum is used, it should be limited to a couple of hours after each change of plastic.  I make prepolymerized containers to place the tissue on and add the embedding plastic on.  It will keep down the bubbles.  When embedding, the air in the embedding container with the liquid plastic will need to be flushed out with C02 (easiest to use), you can also use gaseous nitrogen.  It takes one to two days for complete polymerization in the freezer. 
Things that can cause problems with polymerization:
Containers not flushed enough that have oxygen in them will not fully polymerize. I place the flushed embedding containers in a bell jar, flush jar with CO2 and seal.  This goes in the freezer.  
Humid conditions can cause poorly polymerization ---some hard--some still liquid.  Usually have to replace the embedding plastic, flush with CO2 and try again. You can use drierite in the bottom to help this.
 
After the blocks are hard, the plastic is trimmed a little to the block size I want to use in the microtome.  I wet cut 4 micron sections  and place them on a chrome-gelatin coated slide with a couple of drops of 70% alcohol, on a slide warmer.  The section flattens out, I put a piece of plastic on it, gently roll with a small wall paper roller.  when I get them done I clamp them with c-clamps.  It is good idea to have a small piece of wood and extra glass slide on each end.  It prevents slides from breaking.  They get placed in the oven overnight.  The clamps removed, plastic removed, they are ready to deplasticize with xylene and ready for staining.  You may want to deplasticize with other agents such as 2-methoxyethyl acetate for immunostaining.  Your procedure may give you more info for immunostaining.  I remember doing Brdu with success, however, we would get a halo effect around the possitive cells, and I could not find anyone that had a solution to this.  I did find that
 certain antibody diluents and blockers were a key in the success of this.
 
I hope this helps. 
Nancy
MPI Research
Mattawan, MI