1.	Fix for 20 min with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). Alternatively, cells may be fixed in 100% methanol at -20C for 3 minutes. If methanol fixation is used, skip to step 4.
2.	Wash three times with PBS (pH 7.4).
3.	Permeabilize cells with in 0.2% Triton X-100 plus 1% normal serum in PBS/pH 7.3 for 5 minutes on ice.
4.	Block for 1 hour with 10% normal serum with 1% BSA-PBS.
5.	Incubate for 1-2 hour with primary antibody diluted in 1% BSA-PBS at room temperature.
6.	Wash three times with PBS.
7.	Incubate for 1 hour with secondary antibody diluted in 1% BSA-PBS.
8.	Wash three times with PBS.
9.	Visualize antibody distribution with a microscope.
10.	Store slides at 4°C for future use. *

The above information is only intended as a guide. The research should determine what protocol best meets their needs. Please follow safe laboratory procedures.