1. Cells to be examined
2. 6-well plate
3. Fisherbrand* Superfrost* Plus slides (Fisher Scientific 12-550-15 )
4. Cover slips (Fisher Scientific 12-545-85)
5. Cell culture medium and additives
6. Trypsin-EDTA solution
7. PBS, 4^oC
8. 10% TCA (for cell surface antigens) and 0.1% Triton X-100 (for cytoplasmic antigens), 4^oC
9. Primary antibody, ~ 5-10ug/ml
10. Secondary antibody - florochrome conjugate specific to the source species of primary antibody
11. ProLong Antifade Kits (Invitrogen/Molecular Probes)
12. Nail polish

Procedure

1. Trypsinize cells to get cell suspension in culture medium. Count cell density if needed.
2. Sterilize cover slips in 70% ethanol and dry. Put in 6-well plates.
3. Add cell suspension in the well. Incubate at 37^oC with 5% CO₂ until the cells reach to proper density.
4. Cool cells on ice. Pipet off culture medium and wash in 4°C PBS. Pipet off PBS.
5. If cell surface antigens are being studied, fix 30 min in 10% TCA fixative on ice. If cytoplasmic antigens are being studied, fix 30 min in 10% TCA containing 0.1 % Triton X-100 on ice.
6. Pipet off fixative and wash 3 times in 4°C PBS (5 min/wash). Microcentrifuge diluted primary antibody 2 min at 13,500 x g, 4°C.
7. Layer primary antibody into dish such that cells are just covered and incubate 1 hr at 4°C. Wash four times in 4°C PBS (5 min/wash).
8. Microcentrifuge diluted secondary antibody 2 min at 13,500 x g, 4°C.
9. Layer secondary antibody into dish and incubate 1 hr at 4°C. Wash four times in 4°C PBS.
10. Rinse briefly with H2O and let dry.
11. Mount onto slide with antifade mounting medium. Seal with nail polish.
12. Check under fluenrescent microscope.