Hey Folks,
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.
Thanks,
Caroline
_______________________________________________
Histonet mailing list
Histonet@lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet
|