Immunofluorescence (IF/ICC)

Immunofluorescence Protocol

Visualization of target proteins in cells using fluorescently labeled antibodies.

Procedure

1. Seed cells on coverslips and grow to desired confluency
2. Fix with 4% paraformaldehyde, 15 min at RT
3. Permeabilize with 0.1% Triton X-100/PBS, 10 min
4. Block with 5% normal serum/1% BSA/PBS, 30 min at RT
5. Incubate with primary antibody (1:100-1:500) in blocking buffer, 1 hour at RT or overnight at 4°C
6. Wash 3×5 min with PBS
7. Incubate with fluorescent secondary antibody (1:500) in blocking buffer, 1 hour at RT (protect from light)
8. Wash 3×5 min with PBS
9. Counterstain nuclei with DAPI (1 μg/mL), 5 min
10. Wash 1×5 min with PBS
11. Mount with anti-fade mounting medium
12. Image using fluorescence microscope