So I want to have my cake and eat it too. I have injected a rat with
a virus that will express EGFP. Ideally I'd like to collect the
fresh brain, cut off thick floating sections, enough to get a feel
for the spread of the virus, and possible punch out the EGFP area to
get mRNA and/or protein for qPCR and western blot. Are there any
suggestions for doing this? I have access to cryostats, sliding
microtomes, and brain molds. Cutting fresh brain is difficult, so
I'd like to freeze it to section on the sliding microtome, collect a
floating section, and visualize under a fluorescent dissecting
microscope. Are there any issues with doing this? How should I
freeze the brain, and how will this affect mRNA and protein levels?
How difficult are unfixed floating sections to deal with? Finally,
can I take some sections and fix by immersion in PFA so that I can
store for longer periods of time.
Any suggestions would be appreciated. I've had very good luck
working with perfused/fixed tissue, and would like to continue with
it, but it doesn't seem possible with the mRNA/protein endpoints.
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