What is the best/optimal way to freeze the rat brain - liquid nitrogen, CO2, isoamyl alcohol? When you take out brain from 30% sucrose, what is the next step?
In our lab, after cryoprotection with 30% sucrose we embed the specimen in OCT medium (Tissue Tek or equivalent) and then freeze the block in the vapour phase of liquid nitrogen.
I strongly agree Jayaraj. But I'm used to freezing the rat brains in dry ice and keep then in -80C freezer, until sectioning in sliding microtome. The immunostainning works greatly.
I always used Isopentane filled vials for freezing tissue - then shock freezing in liquid nitrogen. You can then later embedd it (or part of it) in OTC and it's easier to arrange it properly for cutting ...
It depends what you are doing next. If we are moving to frozen sectioning on a sliding microtome, the brain is removed from the cryoprotectant, blocked in any necessary manner, and the bottom side coated with OCT or Tissue Tek. It is then frozen on a stage pre-cooled/frozen with dry ice, and dry ice powder is heaped over the brain. After freezing is complete, we section the brain.
If we are doing cryostat sectioning, then the brain is removed from cryoprotectant, immersed in Peel-Away molds filled with OCT, oriented properly, and then half immersed (not allowing the top to be submerged) in dry ice cooled isopentane. The frozen blocks are left in the molds, wrapped first with parafilm and then a layer of aluminum foil, and stored at -80C until sectioned. We have had troubles with LN2 freezing in the past, most involving fracturing of the brain, so we avoid that method.
Following your question, the next step after 30% sucrose (usually overnight. If perfusion was properly done, the brain will sink in 10-16 h), is to section the brain and keep the sections in 0.1M PBS + 0.1% azyde (if free-floating method) at 4 C... proceeding your staining as soon possible, preferentially the next day. Avoid to keep the whole brains in -80 C freezer for a long time, this is an emergencial tip when you have a lot of brains (~15 or more) and is impossible to cut them in the same or in a couple of days.
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