I need to Postfix mice brains. I slice the brains using microtome at 40 microns. These are Postnatal days 7, 11, 14, and 21. I am using half the brain for other analysis, so I need to fresh freeze it while the other half is fixed.
What should be the best protocol to follow?
We immediately place isolated pup brains in ice-cold 4% PFA and after overnight fixation incubate in 30% sucrose for 48h. It is an advantage that you are working with half brains, nevertheless, check the bigger brains if the overnight PFA was enough for adequate fixation. You might have to adjust length of PFA incubation.
For post-fixing the Postnatal day 21-, 14-, 11- & 7-day-old brains, I usually leave them 2-3 days in ice-cold 4% PFA. Leave it in 30% sucrose until I freeze them. I slice sagittal sections on the microtome. How old are the pups that you use? Is there any tearing/dissolving of the tissue you see while trying to slice them? How do you slide them?
Hello, you can easily post-fix by leaving them in PFA 4% pH7.4 for 24hr to 48hr. Change the PFA to PBS + 20% or 30% sucrose. Pups brains are permeable enough so a post-fixation this long will be enough.
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