Cell monolayers for STORM

  1. Wash cells in PBS
  2. Plate cells in #1.5 glass-bottomed dishes to be 30-60% confluent

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

  1. Remove the o/n media
  2. Add approx. 600µl PFA 3.2% in PBS, incubate at RT for 15 mins
  3. Wash 3x with PBS
  4. Add ≈600µl permeabilization buffer per well, incubate for 5 mins at RT
  5. Wash carefully 3x with PBS
  6. Add ≈600µl blocking buffer per well, incubate for 30 mins at RT
  7. Add primary Abs in blocking buffer, incubate for 30 mins at RT, or at 4°C o/n in MIST tray
  8. Wash 4x with PBS, last wash incubate for 5 mins
  9. Remove the PBS and add secondary abs in blocking buffer
  10. Incubate at RT in the dark for 30 mins, or in 4°C o/n in MIST tray
  11. Wash 3x with PBS
  12. Store in PBS wrapped in parafilm at 4°C until imaging
Permabilization Buffer: per 100ml
NP-40 1 ml
PBS 99ml
Blocking Buffer: per 100ml
NGS/NDS5 ml
Brij detergent 10%1 ml
PBS 94ml
Primary conc.Secondary conc.
mab3211 1:50Alexa 647 1:1000
H4K20ME3 1:50Alexa 488 1:100