Methods and Techniques Discussion => Immunocytochemistry (ICC) => Topic started by: MJoanna on March 11, 2013, 09:23:52 PM

Title: ICC on mouse ESCs
Post by: MJoanna on March 11, 2013, 09:23:52 PM
Hello all,

  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.

Thanks!
Joanna
Title: Re: ICC on mouse ESCs
Post by: gula on March 12, 2013, 11:34:33 AM
I'm no expert in cell-handling, but do you think, there could be an osmotic stress from the ICC reagens on your cells?

Perhaps longer fixation brings more stability.
Gula