I differentiated human neural progenitor cells into neurons. These cells were relatively fragile and were difficult to attach on the cell surface. I have to immunostain these cells, thus I was wondering if the temperature of PFA may affect the immunostaining results obtained from the cells.
Hello I do work with direct reprogramming of neuron cells from fibroblast and immunostain these according to my experiment. I fixed the cells with both in 4 degree and room temperature. Even though both worked nicely but i prefer to do PFA in room temperature as 4 degree PFA can sometimes causes shrinking of the cells.
Hope that help.
Masuma
Hi Saujanya Rajbhandari,
fixation at lower temperatures is normally done with tissues or whole organisms because it preserves the structure better during long fixation times (overnight or longer). For fixation of cell culture dishes, room temperature is usually fine.
Best,
Lukas
Sebastian Schmitt
, Thank you very much for your comment. I understand the adverse impact of keeping the PFA in a cell incubator.
the question was -whether temperature may impact ICC results. It may or may not, depending on the antigen, the epitope and your antibody (monoclonal, polyclonal). Some people fix cells for 1 min in 1% PFA to get ICC to work because overfixation causes loss of ICC signal. A quick answer -if your antibody and the antigen are not impacted by longer fixation then staining at room temperature, say 15 min, 4% PFA, is ok. If your antibody is not working well to begin with, then try to reduce the length/strength of fixation by either making it shorter, or/and diluting 4% PFA down to 1% or even 0.5%, or/and fixing at 4C or on ice. As one person replied earlier - this is a chemical reaction, and it goes faster at room temp. Also, if you are comparing results from several different experiments done at different time - pick one way you are fixing cells for ICC and stick to it, otherwise it may be difficult to compare the signal because of the reason mentioned above (over-fixed cells may produce weak/no signal with SOME antibodies). Polyclonal antibodies have higher chance of working better even with overfixed cells because they can detect several epitopes. PFA (reversible fixative) gradually converts to glutaraldehyde, irreversible cross-linker, and room temp and light accelerate this process, yet 10-15 min is a short time. If you are fixing for a long time and at room temp then it can make a difference.
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