Is it vital to fix cells in all ICC protocols? I am staining for a cell surface receptor, CD163, and plan on imaging immediately after the staining protocol and doing all incubations at room temperature.
Is it okay to skip the fixation step or is it recommended to keep it?
Thanks!
well it is highly recommended to fix the cells, once i tried without fixiing and i lose nearly the 50% of my sample before staining. so i recommended to keep fixing
Yes, it is important to go throw fixation before you start your staining, but take care you may need to do some optimization firstly for the type and the concentration of the fixative and also pay attention to the time you should use as this may affect the following steps.
If you are not looking for something conformation specific or the receptors dynamic changes, it is important to fix the cells to avoid loss of cells as well as the loss of antigen integrity throughout the procedure. Only thing is that look for the appropriate fixative and the timing to avoid false positive results or no results.
Many cell-surface CD antigens don't require fixation; you can label the cells live with your antibody and image right away, for flow cytometry. Fixation, though, will give you more time. I am not certain, however, if CD163 is one of those. It would be best to consult with the Tech Support of the company you purchased the primary antibody from.
fixation is very important, i recommend to you: first do a smear on a charged glass-slide, use a drop of 100 microliters of your cell suspension, wait to dry at room temperature, then fix your cells with acetone 10 minutes at 4ºC, and wash with PBS TRITON O.5 %.
NOTE:
"But, some antibodies works without fix the cells, try it too"
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