[Histonet] Basic staining questions

From:"Keith Mc Quillan"


I am pretty new to staining (as in i have only ever tried it once) and would
appreciate any help you can give me with regard to processing my tissue and
staining. I will be processing mouse brains in the coming weeks with the
intention of staining for amyloid, and want to check about the correct
method for fixing this tissue. In our lab, we have always snap frozen tissue
in OCT directly after taking it, no sucrose gradients or aldehydes
etc.However, i have noticed from reading the literature that most labs seem
to fix the tissue in 4% paraformaldehyde before fixing in paraffin. Does it
make any difference as to which protocol is used? I am reluctant to change
unless i have good reason to. I am planning on perfusing the animals with
PBS before  taking half the brain for immunohistochemistry (in OCT) and the
other half for ELISA/RNA etc and so can't perfuse with PFA.

My other question relates to the staining for amyloid beta itself. I have
noticed that a number of protocols incorporate a step to treat the sections
with 70% formic acid prior to staining. Is this neccessary for the staining
of amyloid, and if so, what advantages does it offer.

Any help anyone can provide me with would be greatly appreciated

Keith Mc Quillan
Trinity College Dublin
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