Good day !
I did a pilot experiment to quantify c-fos expression in amygdala after fear exposure. I had a control group with no exposure to the threat.
The c-fos protocol I used was one from our lab which recommend 3 days incubation at +4 degrees in primary antibody (rabbit, Cell signaling) and overnight incubation in secondary Alexa 488 (goat anti-rabbit). The staining is beautiful with a lot of cells. Problem : I have the feeling that it is actually too much. First, I do not see any difference between my test group and my control group while we know through literature that we should see a difference ! And secondly, I do see a lot of staining a bit everywhere (Cortex, hippocampus, thalamus, etc). Is it possible that with a too long protocol we do not stain only activated cells ?
I am now trying a shorter protocol (1 day in primary, 2 hrs in secondary). I do see staining but less cells. Now I am confused, which one should I go for ?
I work with mice brains, free-floating sections, 80 um thickness and do a permeabilisation protocol and blockage with Triton and BSA-FBS.
Thanks for your help !
Hi Noemie Mermet-Joret ,
I agree that the protocol you ran was too long. In three days of staining and incubating and considering the fact that your slices are somewhat thin, it would not be surprising if you see stained cells other than your area of interest, especially in a rather dense area like amygdala, BLA which are close to hippocampal formation.
1 day seems plausible. Though if you would like to have better results, test different temperatures or antibody and staining solution concentrations.
I have only ever run c-fos stainings over-night, i.e. even less than 24 h (using 30 µm sections) and they look fine (granted, I have not used the same primary as you have). I agree with the previous reply, 3 days seems exaggerated.
It is completely normal to see some cfos-positive cells even in controls, particularly in cortex and hippocampus, because of regular physiological signaling (unless they were anaesthetized for several hours prior to sacrifice).
- Badges
- Science topic