A query regarding post-fixation

[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.

[MT Scientist]

I would guess it is the freezing at -80C and thawing prior to cryosectioning that is causing damage.

[Cardio]

Also the longer the fixation the higher the autoflouresence can be. Typically you shouldn't freeze thaw samples like Mt is saying.

She is also washing out the PFA and you are not so go with the protocol that works.