I am working on pure pDC populaion of human origin and I need to detect phosphorylation of AKT after stimulation with Imiquimod. I fix the cells after stimualtion with paraformaldehyde. Can I store these fixed cells and permeabilize afterwards? If yes, what should be the storage procedure and temperature for fixed cells?
i routinely fix and extract coverslip of different cell lines with PFA/Triton/FCS in BRB80 buffer at 37° 20-30min. upon fixation, I keep the CS in PBS with added Na-azide 0,05%...it is fine for my needs..of course you have to validate this for your need.
I fixed my raft section and stored the slides at -20 for several days. It is no problem to continue with what you proposed. No experience with your DC though.
By the way, what's the protocol for detection of phospho-AKT? It is said that appropriate blocking buffer or antibody buffer is needed for good results.
I don't think it is a good idea to store cells in PFA for a long time. Long fixation leads to more protein cross linking
Usually I've left them for 24h (at most), usually It affects signal of surface markers beyond that. I do not recommend it anyway. The best is to run them immediately after, or just leave them Overnight and run them in the morning
@ Carlos..can I fix with PFA, wash and then store at 4 degree for max 24 hours?
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