Plate 1-5x104 cells in a total of 500 µl medium per well in an 8-well chamber slide (corresponds to appr. 10-50 µl cells from a confluent 10-cm plate taken up in 10 ml, or a 3.5-cm plate taken up in 2 ml).
Incubate over night.
Be gentle for all subsequent steps. Add liquids slowly above one corner of chamber. Do not let cells dry out.
Remove medium.
Fix cells by incubation for 20 min at room temperature with 100 µl of freshly prepared 3-4% paraformaldehyde in PBS (dissolve in boiling PBS, and cool to RT).
Wash cells twice with 500 µl PBS for 5 min.
Permeabilize cells by incubation at RT for 15 min with PBS/1% goat serum/0.5% Triton X-100.
Wash three times with 500 µl PBS/1% goat serum.
Incubate for one hour at RT with primary antibody diluted in 100 µl PBS/1% goat serum. (a-FLAG mAb or a-myc mAb: 10 µg/ml; rabbit sera: 1:100-1:1000 dilutions).
Wash twice with 500 µl PBS/1% goat serum.
Incubate for one hour at RT with fluorescent anti-IgG antibody diluted in 100 µl PBS/1% goat serum. (Texas Red labeled anti-mouse or anti-rabbit IgG: 2-4 µg/ml).
Remove medium.
Incubate for 10 minutes with 1 µg/ml DAPI in 100 µl PBS/1% goat serum.
Wash three times with 500 µl PBS/1% goat serum.
Wash once with 500 µl water.
Remove top of chamber slide. Remove all water and air-dry briefly.
Add two drops of Vectashield.
Mount coverslip (avoid bubbles) and seal with nail polish.