fixation and immunostaining of neurons after live-cell imaging - (Apr/18/2012 )

Hello,

I have been running an experiment that involves imaging axonal mitochondria using a transiently transfected fluorescent protein. Imaging is done every 15s for one hour. Recently, my PI has asked me to fix the cells after the time lapse and immunostain for a particular protein. I tried it (using 4% PFA), but I couldn't find the cell I had been previously imaging, and the cells in the dish appeared to have shifted. I'm quickly starting to think that this might be futile effort, but does anyone have experience doing anything like this?

Thank you so much for reading.

Hi

I performed some time ago experiments including live neurons. First thing is that, in my case, the antigen I was trying to find disappeared after fixation so I did the ICC live. I incubated the antibody before fixation, 1h in the incubator. After that I fixed the neurons (in my case from the hippocampus) with 2% PFA and then proceed with the rest of staining. Don't ask me why but the ICC only worked with 2% fixation and not with 4%. So, maybe after finishing the time-lapse you can apply the primary ab, then fix lightly and see what happens. If it works, please let me know. I'm curious to see if it's the same with all antigens in neurons or just with mine.

Good luck