Cell monolayers

  1. Wash cells in PBS
  2. UV irradiation and EtOH wash of coverslips, wash away EtOH with PBS or media before adding cells
  3. Plate cells in 6-well plates with coverslips to be 30-60% confluent

The following day cells should be ready (60-90% confluent)

  1. Remove the o/n media wash the cells 3x with cold PBS pH 7.4
  2. Add approx. 200µl PFA 3.2% (dissolved in PBS) to cover, incubate at RT for 20 minutes.
  3. Wash 3x with PBS
  4. Permeabilize cells in PBS containing 1% NP-40 for 5 minutes (with approx. 200µl per well)
  5. Wash carefully 3x with PBS to rinse all NP-40 away
  6. Block for minimum 30 minutes at RT, with blocking buffer (5% NGS or NDS and 0.1% Birj in PBS.
  7. Add primary Abs diluted in blocking buffer, spin down and apply the supernatant.
  8. Incubate primary ab (1:5 mab3211) for 30 minutes at RT, or at 4°C o/n, in MIST tray.
  9. Wash 4x with PBS, last wash incubate for 5 minutes.
  10. Remove the PBS and add secondary abs that have been diluted in blocking buffer (1:100 Alexa 555).
  11. Incubate RT in the dark for 30 minutes, or in 4°C o/n in MIST tray
  12. Wash 3x with PBS
  13. Dip the coverslip in water.
  14. Mount the coverslip on a glass slide.
  15. wick excess fluid by touching coverlsip edge to paper, dry at RT for 30 minutes.
  16. Clean the microscope slide with 95% EtOH and wipe dry.
  17. Add a small (approx 6 µl for small round coverslips) drop of mounting media to the slide.
  18. Place the coverslip on the mounting media cell-side down, allow the mounting media to dry for approximately an hour.