Hey everyone,
what risk could there be on cells that have been left in 4% formaldehyde for over an hour?
Normally I would leave the cells in this solution for 15 minutes but I left them for an hour.
This preparation was to fix the cells for flow cytometry analysis.
Thank you for your answers.
PFA is a small molecule that rapidly infiltrates cells. For cells recommended time is 15 minutes, over fixation can renders some antigens irretrievable and increase chances of autofluorescence.
You could run an unstain prior to antibody incubation.
Your answer makes sense with the results that I got. Actually I have autofluorrescence on the cells.
Increasing incubation time will render your antigens inaccessible to antibodies as over-fixation can modify the amino acids that are part of an epitope and can block an antibody from binding to it. Formaldehyde binds to side chains of cysteine, histidine, lysine, arginine...
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