1. Plate cells in chosen chamber slides and grow cells 1-2 days in appropriate medium. Cells need to attach strongly to plate.
IMPORTANT! Some cell lines will need special coating i.e. poly-lysine of the chamber slides to aid in cell attachment. The specific type of coating needs to be determined empirically as it varies between chamber types and/or cell lines.
2. Wash cells 2-3 times with ice-cold PBS.
3. Fix cells by adding paraformaldehyde (PFA) 1- 4% in PBS 10 min at room temperature.
Note: the optimal PFA dilution depends on the cell type and should be established experimentally.
4. Wash cells 2-3 times with ice-cold PBS.
5. Permeabilize cell membranes by adding saponin assay buffer (PBS + 2% BSA + 0.1% saponin) 10 min at room temperature.
6. Block unspecific binding sites with 5% goat serum in saponin assay buffer for 15 min at room temperature.
7. Add primary antibody at the appropriate dilution in saponin assay buffer and incubate for 1-2 hr at 4°C.
Note: If using fluorescent labeled antibody, skip to step 10.
8. Wash cells 3 times with saponin assay buffer.
9. Add fluorescently-labeled secondary antibody at the appropriate dilution in saponin assay buffer and incubate 1 hr at 4°C protected from light.
10. Wash 3-5 times with saponin assay buffer and drain well. Add buffer to cover cells and proceed to the microscope.