IF/ICC Immunocytochemistry and Immunofluorescence

 

  1. Reagents
  • PBS 1x, autoclaved
  • PFA 4% (stock 16%, #15710, Electron microscopy sciences)
  • Perblock: 1% BSA, 0.05% saponin in PBS; keep in aliquots at -20°C
  • DAPI (1 ug/ml)
  • Moiowol
  1. Fix cells
  • Aspirate cell medium and cells that have not adhered
  • Wash 3x with PBS
  • Fix cells with 300 µl of PFA 4% (or cold methanol), 10 min., RT
  • Wash 3x with PBS (cells can be kept at 4°C for a few days)
  1. Perform immunofluorescence microscopy
  • Remove PBS/Block and permeabilize cells with 300 µl Perblock, 30min, RT
  • Add primary antibody diluted in Perblock (test 2-3 dilutions 1/50-1/500; 50 µl/sample); incubate 1 h, RT, humidified chamber, in parafilm drops
  • Wash 3x with PBS
  • Add secondary antibody diluted in Perblock (50 µl/sample); incubate 30 min, RT, humidified chamber, in parafilm drops
  • Wash 3x with PBS
  • Stain with 300 µl DAPI, ready to use for 5 min, RT
  • Wash 3x with PBS
  • Mount coverslips with Mowiol; let dry overnight at RT
  • Keep at 4°C