Jul 15, 2024
Cryoprotection of mouse brain tissue
- 1UCSD;
- 2University of California, San Diego
Protocol Citation: Lauren C. Faget, Thomas Hnasko 2024. Cryoprotection of mouse brain tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5x9jg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 15, 2024
Last Modified: July 15, 2024
Protocol Integer ID: 103430
Abstract
Fixed brains are submerged in 30% sucrose to cryoprotect prior to freezing and cutting cryosections.
Safety warnings
Dispose of fixative-contaminated sucrose and isopentane as hazardous waste or according to your intuitional guidelines
Before start
Following perfusion and post-fixation in 4% ice-cold PFA (for 2 hrs, overnight, or as appropriate for intended use):
Procedure
Procedure
2d 0h 0m 15s
Make 30% sucrose solution by dissolving 30 g of sucrose (BP220-1, Fisher) in 100 mL of 1X PBS (BP3991, Fisher) 7.4
Fill tube (20 mL glass scintillation vials or 15 mL conical centrifuge tubes are classically used) with ~ 10 mL of sucrose and transfer fixed brain tissue into tube, gently mix by inverting tube several times.
Incubate at 4 °C until brain sinks (typically ~48:00:00 ).
2d
Prepare superchilled isopentane (2-methylbutane, M1246, Spectrum) by pouring ~30 mL 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 ~00:00:15 until completely white and bubbling stops.
15s
Transfer to storage-tube that has been pre-labeled and superchilled on dry ice.
Transfer to -80 °C freezer for long-term storage.