Immunofluorescence - Problems with fixation (Jan/07/2010 )
I have been trying to fix epididymal cells on glass coverslips and view secreted or surface proteins. I have tried different protocols for fixation. The cells seem to grow fine on the coverslips and fix well after formaldehyde fixation. However, we do not want to permeabilize the cells - so we do not use any permeabilizing agent such as triton or methanol. But we still continue to damage cells somehow after fixation.
We tried to use live cells wihthout fixing them, but these cells come off the coverslip somewhere in between the entire procedure - I think its mainly during the washes between antibodies.
Did anybody have a similar problem with fixation where the cells are damaged/permeabilized when they are not ideally supposed to be! I do not want the antibodies to go inside the cells as all we are interested in looking at are the surface and secreted proteins.
What percentage of paraform are you using?
Try to use PFA (paraformaldehyde) 4%!!
Do you use coated coverslips? If your coverslips were coated with poly-L-lysine the live cells should stick no problem and remain attached for the washing procedures
use PBS with calcium and magnesium, if you want avoid cells detaching from coverslips.
If you haven't already tried this: Wash cells, then add primary antibodies to the cells and incubate in primary at 4C, before fixing them.