Cryoprotection of mouse brain tissue
Lauren C. Faget, Thomas Hnasko
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
Fixed brains are submerged in 30% sucrose to cryoprotect prior to freezing and cutting cryosections.
Before start
Following perfusion and post-fixation in 4% ice-cold PFA (for 2 hrs, overnight, or as appropriate for intended use):
Steps
Procedure
Make 30% sucrose solution by dissolving 30g of sucrose (BP220-1, Fisher) in 100mL of 1X PBS (BP3991, Fisher) 7.4
Fill tube (20 mL glass scintillation vials or 15 mL conical centrifuge tubes are classically used) with ~ 10mL of sucrose and transfer fixed brain tissue into tube, gently mix by inverting tube several times.
Incubate at 4°Cuntil brain sinks (typically ~48h 0m 0s).
Prepare superchilled isopentane (2-methylbutane, M1246, Spectrum) by pouring ~30mL into 50 ml bottle or beaker. Carefully position beaker on dry ice in a stable manner. Wait until isopentane temperature reaches -30°C before proceeding to flash freezing.
Rinse brain with chilled 1X PBS.
Dry brain on paper towel by rolling it and gently pressing against paper towel.
Use long-handled forceps to transfer brain to superchilled isopentane. Let brain freeze for ~0h 0m 15s until completely white and bubbling stops.
Transfer to storage-tube that has been pre-labeled and superchilled on dry ice.
Transfer to -80°C freezer for long-term storage.
