I perfused mice intracardially with 2%paraformaldehyde +2%glutaraldehyde, and stored in similar medium for 3-4months. we washed brain twice or thrise with PBS solution before cryosectionig procedure at -20 degree.
Impressions: sections are not uniform, lot of micro holes and cracks (sig of dehydration) visible in cresyl violet staining on 7-8 micron section.
Hi! I agree with the last post. You need to equilibrate your sample before freezing. I use to do it as following. 15% Glucose in PBS 4h and 30% overnight at 40ºC. After this embed in OCT, freeze in dry ice and store at -80ºC. Then, the cut is perfect. Good luck!
As I read your protocol, then I have to conclude that the problem is the fixative and the storage for a long period in the fixation solution. Why not using an intracardially fixation procedure with 4% paraformaldehyde in PBS (with or without a short post-fixation) and then proceed (as described in the other posts) for embedding in OCT?
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