As one might have already noticed in a recent post, I’ve come across a need to make a brain matrix. I’m going to post the recipe for what I plan to do but be forewarned, as of yet this is untested. (If you try it let me know how it goes.)
Supplies:
- 96-well plate, preferably one used in freezing samples so the cells hold about 1ml and come apart from the plate
- 96 wedges from a real brain, about 1mm x 1mm x 0.5-1mm
- agarose
- lecithin
- Bovine serum albumin (BSA)
- 0.1M sodium cacodylate buffer (or whatever buffer you prefer but be aware of contamination and consider using a little sodium azide – very hazardous stuff!!! BE CAREFUL!)
- All kinds of crazy stains and chemicals
First off, we’ll need enough agarose to cover the brain wedge to the thickness of the shortest radius between the center of a real brain and the surface (somewhere around 3mm). According to this chart, a well is about 10mm deep and holds 360ul. I plan to use something more like this but the diameter should be about the same. So chances are 200ul per well should be more than enough matrix medium. If I make 20mls it should be more than enough.
Incidentally, before you get started, measure exactly how much of the mix you’ll need to add to bring the height to 3mm.
Melt the agarose in water. We’ll need to melt it at a higher concentration than we need because we’re going to be adding BSA and lecithin.
0.48g of agarose in 20mls of water will give a 2.4% gel. This is four times the concentration of what we’ll need but it should still melt just fine. So weigh out 0.12g of agarose in 5mls of water. Microwave it very briefly (short increments) since it’s such a small volume. If water escapes, add warm water to reconstitute to 5ml.
Since BSA shouldn’t be microwaved, dissolve it in a separate container (in water) for a final concentration of twice what we’ll want to end up with. As mentioned in my post, we’ll shoot for 13% protein in this matrix. 13g in 100ml is 13%. In 10ml, 1.3g We’ll make 10mls but have double the concentration so it comes to what we want when we increase the volume to 20ml. Dissolve 2.6g BSA in 10ml water.
Finally, the lecithin. We’ll want to add 20% of the entire volume as fats from lecithin. That means we want 4mls of lecithin or 4g/20ml agarose. When everything is warm and melted (but not too hot to destroy the BSA), add the following together:
- 10ml 26% BSA
- 5ml 2.4% agarose
- 4ml dissolved lecithin (or 4g of dry)
- Warm water to 20ml
pH the solution before adding the last ml of water. A pH of 7.4 is physiologically valid but since I’ve never done this I have no idea how the pH will turn out. Maybe add a little acid, maybe a pellet of NaOH. Either way, you won’t have much volume to play with. Then top it off the rest of the way to 20ml with water. (If the water is too cool it will cause the agarose to become solid.)
Find a deep 96-well plate and take 1mm wedges from a real brain to put in the bottom of each well. Dip each wedge in the matrix mix (that’s fun to say) before putting it in the bottom of the well. This is for a couple of reasons – we want to avoid bubbles as much as possible and this will help the brain wedge be as cohesive with the matrix as possible. Dipping and placing will give the brain wedge time to cool and harden and will make it more likely to stay where we put it (provided we don’t blast it when we pour in the still-warm liquid matrix mix).
Once all the wedges are placed, add enough of the matrix mix to reach a uniform height of 3mm. Let it solidify. Agarose is amazing. It almost always takes my mixtures 45 minutes to become solid (faster if you cool it by putting it in the fridge or the freezer briefly).
Cover each well in a little buffer so the agarose doesn’t dry out. Sodium cacodylate is the buffer I’ll be using for two reasons: 1) bacteria doesn’t like to live in it because of the high arsenic content, and 2) it’s a good buffer for many electron microscopy recipes and that’s where I want to go with this.
I hope this works.
