[Histonet] Polymerizing BIG methyl methacrylate blocks

[Jack Ratliff]

Paul,
 
I can tell you from many years of experience that you have to be very careful when polymerizing large amounts of MMA. As you probably know, it is an exothermic reaction that can and WILL generate a significant amount of heat depending upon the percentage/amount of catalyst you use, the size and density of the tissue, total volume used, and the temperature at which the solution is left at to polymerize. If you do not control the polymerization, it will reach its v-max too fast which will result in an over-polymerized mess and ruin your tissue.
 
My best advice to you immediately is to take a look at the concentration of catalyst you are using and consider lowering it depending on what you are currently using. I use approximately 0.25% w/w of dry Perkadox-16.  A couple of days before I am ready to embed my tissue, I will make up 1000 mL of MMA + DBP and then add 2.5 g of Perkadox. I do this at the end of the working day and let it stir lightly overnight (22.2 - 22.5 C) in the hood at room temperature to 'ripen'. The solution will change from clear to a shade of yellow (lighter or darker) that is somewhat proportional to what room temperature means in your lab.
 
The next piece of advice is how you control the rate of polymerization. The following morning I will put the solution into the fridge overnight to slow the reaction. The very next day, I bring the solution out of the fridge and allow to warm to room temperature. This is to avoid any potential for moisture as you don't want a cooled MMA solution to have the chance to meet with a room temperature tissue and create ANY chance for moisture. It could make your final polymerized block cloudy instead of clear. I then add my embedding solution to the embedding mold w/ pre-polymerized base layer and drop in the tissue. With the lid off, I then place the specimen, within its embedding container, under vacuum (-18 to -20 inHg) until the end of the day (6-8 hours). Don't worry about orientation at this point because you will attend to it before you leave for the evening. At the end of the day I will put the tissue into the desired orientation, put the lid on, and place it in the fridge overnight. The next morning, I simply bring out my embedding container (lid on) and leave it at room temperature (22.2 - 22.5 C) for the rest of the day and then overnight (if you feel that you can trust room temperature to stay consistent) and repeat as necessary.
 
Basically, I babysit my specimens on a daily basis over the next 5-9 days (depending on the tissue size/density and volume of embedding solution) and alternate between room temperature and the fridge to help evenly polymerize my specimen so that I achieve a nice clear block. Once the block is mostly polymerized and definitely above the level of the tissue with only a very thin or tacky layer on top, I place the container in a waterbath (level of water slightly above the block and fluid level) and leave it at 37 C overnight until the block has polymerized. The next day I will then put the container (without the lid) into an oven at 60 C overnight to fully complete polymerization and evaporate most of the residual MMA smell. Believe it or not, but the block still could be polymerizing even though it is firm to the touch after the waterbath step. Also, this oven step helps to drastically reduce the MMA smell when you go to break free from the mold and handle the block.
 
Large tissue polymerization is really something that you cannot predict routinely because of all the variables involved. However, if you can manage catalyst concentration and your room temperature environment, the time estimation of polymerization for specific tissues (size/density and volume of solution needed) becomes easier to calculate and repeat successfully. Try this out and please let me know if you need any clarification.
 
 
Jack Ratliff
 
 
> Date: Fri, 5 Dec 2008 15:14:07 -0500> From: PMonfils <@t> Lifespan.org> To: histonet <@t> lists.utsouthwestern.edu> Subject: [Histonet] Polymerizing BIG methyl methacrylate blocks> > Does anyone here have experience with large PMMA blocks, about 150 to 200 ml volume each? Specifically, at what temperature do you recommend polymerizing them, and how long should the polymerization take? Any other helpful hints will be appreciated.> > Paul M.> _______________________________________________> Histonet mailing list> Histonet <@t> lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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