General Research Topics > Neuroscience

A query regarding post-fixation

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KLavi:
Hi all,

I am trying to measure c-Fos expression in free floating brain sections using immunofluorescence. For some reason, I don't see the c-Fos in the nucleus. I see it mostly in the cytosol and dendrites. In addition, the signal to noise ratio is pretty bad..

I have managed to focus the problem on the post-fixation process; I have asked a colleague to use her brain sections. Using my staining protocol and same antibody I was able to view an amazing signal, located in the nucleus and with a really good signal to noise ratio.

There are some differences between out post-fixation protocols.
The protocol I use for post-fixation is:
96 hours in 0.01M PBS + 30% sucrose + 1% PFA in 4C.
after the brain has sinked I take it out and freeze the brain in -80C
couple of days \ weeks later - I defreeze it and use the microtom to slice it.

Her post fixation is different than mine. First she keeps the brain in 0.01M PBS + 4% PFA over night, following by a gradient of PBS + sucrose (10% for 24 hours and then 20% for 24 more hours). She does not freeze the brain after the post fixation process, but keeps it in 4C until slicing.


What do you think causes my problem? Which protocol do you use?

Thanks in advance!
Karen.

CanuckPhD:
Your colleague's protocol is very similar to my protocol in which we get good tissue preservation. If you freeze your tissue at -80, often ice crystals form (even with sucrose) and the tissue is damaged. Have you performed a creysl violet on your tissue to look at the morphology?

After the tissue is fixed, degradation of antigen is low at 4C, so you can usually leave the tissue there for weeks before slicing. Of course you have to confirm this with your antigen but for the ones I have used it is fine.

disco volante:
We use a method fairly similar to your colleague's and it has always worked well.  I wonder if the high sucrose concentration in your post-fix solution is mashing up your cell membranes before they are fully fixed (osmotic pressure isn't so much of a problem on fully fixed tissue)

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