Tissue Culture Cell Immunofluorescence
1. Place slide on ice for 5 minutes. While on ice, remove medium and wash with 0.25 ml of ice-cold
PBS. Avoid dislodging cells. Do not allow cell monolayer to dry during steps 1-9. Alternatively,
wash with PBS at room temperature and do fixation at room temperature (for 30 minutes).
2. Fix cells with ice-cold 3% pFA in PBS + 1 mM MgCl2 for 15 minutes or longer on ice. Place slide
at room temperature for 15 minutes.
Prepare fixative by diluting 30% pFA 1/10 into PBS + 1 mM MgCl2 at room temperature. Check
pH of PBS + 3% pFA. Adjust to pH 7.2 with HCl. Chill on ice before use.
Paraformaldehyde 30% (pFA) stock:
A. Add 2.0 g pFA and 5 mls ddH2O to Pyrex tube (16 X 150 mm).
B. Heat slowly with burner to boiling and slowly vortex intermittently.
C. Add 0.1 ml 1M NaOH while vortexing. Place at room temperature for 5 minutes.
D. Spin in IEC for 5 minutes at 2500 rpm at room temperature. Or, do 0.45 Ám filtration.
E. Store 30% pFA stock for no longer than one day at room temperature.
3. Wash cells 3 X 5 minutes with PBS. Add 50 mM NH4Cl to middle wash (dilute a 5M NH4Cl solution
1/100 in PBS). This step and those below are done at room temperature.
4. Permeabilize fixed cells for 5 minutes with 0.25 ml of PBS + 1.0% Triton X-100 (PBS-Tx).
5. Block cells for 5 minutes with 0.25 ml PBS + 1% BSA (PBS-BSA).
6. Dilute primary antibody (1░ Ab) in 0.2 ml of PBS-BSA. Incubate for 1 hour.