Afra

Cell monolayers

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

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