1. Plate cells in chosen chamber slides and grow 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 assay buffer (PBS + 2% BSA + 0.05% NaN3).
3. Add primary antibody at the appropriate dilution in ice-cold assay buffer. Incubate 1 hr at 4°C.
Note: If using fluorescent labeled antibody, skip to step 6.
4. Wash cells 2-3 times with ice-cold assay buffer.
5. Add fluorescently-labeled secondary antibody at the appropriate dilution in ice-cold assay buffer and incubate 1 hr at 4°C protected from light.
6. Wash 3-5 times with assay buffer and drain well. Add buffer to cover cells and proceed to the microscope.