Flash Freezing and Cryosectioning E12.5 Mouse Brain

Summary

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

Protocol

1. Fix tissue in 4% paraformaldehyde in PBS for desired time.
2. Sucrose infuse tissue (cryoprotection)
   1. Make 30% sucrose solution in PBS w/v in 2059 tube.
   2. Rinse tissue 3x in PBS (~ 5 min with rocking).
   3. Place tissue in 30% sucrose solution.  Tissue will not sink.
   4. Place the tissue in 4°C overnight, or until it has sunk.
3. Label appropriate size cryomold with information and orientation.
4. Fill cryomold with O.C.T. (avoid bubbles).
5. Transfer tissue to O.C.T. bath and coat it with O.C.T.
6. Transfer tissue to O.C.T. in cryomold.
7. Orient the tissue under microscope.
8. Pour liquid nitrogen into plastic Petri dish.
9. Quickly and carefully lower the tissue in cryomold into the nitrogen. (do not submerge the top of the cryomold.)
10. When the O.C.T. is solid white, place the frozen tissue into -80°C freezer for storage.
11. Equilibrate tissue to ~20°C for at least 30 min. prior to sectioning.

Materials

Name	Company	Catalog Number	Comments
Tissue-Tek Cryomold	Ted Pella, Inc.	27181
O.C.T.	Ted Pella, Inc.	27050
Sucrose solution			30% sucrose solution in PBS w/v
paraformaldehyde			4% paraformaldehyde in PBS