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
Fix tissue in 4% paraformaldehyde in PBS for desired time. Sucrose infuse tissue (cryoprotection) Make 30% sucrose solution in PBS w/v in 2059 tube. Rinse tissue 3x in PBS (~ 5 min with rocking). Place tissue in 30% sucrose solution. Tissue will not sink. Place the tissue in 4°C overnight, or until it has sunk. Label appropriate size cryomold with information and orientation. Fill cryomold with O.C.T. (avoid bubbles). Transfer tissue to O.C.T. …
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 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 |
Cite This Article
Currle, D. S., Monuki, E. S. Flash Freezing and Cryosectioning E12.5 Mouse Brain. J. Vis. Exp. (4), e198, doi:10.3791/198 (2007).