Hello, want to do co-localization in rats brain by Immunofluorescence. What would be the ideal way. If i want to use fresh sample, is it possible to keep brain at -20/-80 for sometime to solidify & then go for frozen sectioning. Or if i want to preserve sample for some time, what would be ideal way: to use isopropanol+ snap freeze or keep it in IF fluid?
For IF, you need to section the brain and for that you need to freeze it. When you are collecting brain from rat, use cryomold (a plastic like devise) to keep the brain in OCT gel and snap freeze in dry ice. You can store this frozen brain in -80 for long time. For sectioning, take out frozen brain from -80 and use cryosection machine to cut them in to your desired thickness (normally 5 micron) at -20 to -30 C and fix sections immediately. Hope this helps.
Thanks Subrata Pore and Lukas. I used 5um coronal sections from fresh tissue using OCT. I am getting a lot of auto fluorescence even i used H2O2 and blocked with serum. Can anyone suggest me anything?
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