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Immunostaining Protocol

  • Wash cells once briefly with PBS and fix for 20min with 4% paraformaldehyde (PFA) in PBS (we use Electron Microscopy Sciences Cat#15710 diluted to 4%)
  • Wash 3x10min with PBS at RT
  • Saturate non-specific sites with 10% normal goat serum (NGS) in PBS for 40min at RT. For intracellular epitopes include 0.3% TX-100 to permeabilize.
  • Wash 3x5min with PBS at RT
  • Dilute the antibodies in a fresh solution of 5% NGS in PBS and incubate for 1hr at RT. Common antibodies are listed below.
  • Wash 3x5min with PBS at RT
  • Dilute appropriate secondary antibody in 5% NGS/PBS and incubate cells for 1hr at RT
  • Wash 3x5min at RT
  • Either mount in DAPI-containing Vectashield or incubate with 0.2ug/ml bisbenzimide in PBS for 10min, wash and mount in AquaPolymount. If cells were not previously peremeabilized, do so by incubating for 5min with ice-cold methanol. Wash x 2 before adding dapi or bis-b.
Antibody Dilution Source Catalog Number
SSEA1 1:100 Santa Cruz sc-21702
SSEA4 1:100 Santa Cruz sc-21704
Tra-1-60 1:100 Kind gift of Peter Andrews, Sheffield University
Tra-1-81 1:100 Kind gift of Peter Andrews, Sheffield University
Oct-4 1:20 Santa Cruz sc-5279
MDM2 1:100 Santa Cruz sc-965
Nanog 1:100 ReproCELL Inc/Cosmo Bio Co Ltd RCAB0004P-F