I am trying to characterize some mESC lines with the stem cell marker panel from Abcam (Oct4, Sox2, Nanog, SSEA1). I am having some issues with the protocol. It seems that after fixation with 4% PFA my cell morphology is distorted and by the end of the protocol after the final washes I lose all my ESCs. I only see some of the feeder MEFs remaining. Any tips or a protocol you know works for these cells?
I coated #1.5 coverslips with 0.1% gelatin. Grew cells, washed with 1x PBS 3x, fiixed with 4%PFA 20m, washed again, permeablized with tritonx 100. blocked with NGS/BSA/tween O/N. washed again, primary Ab in Dako dilutent O/N. washed again. secondary Ab 1hr RT in dark. washed again, mounted with either vectashield or antifade.