ICC for Adherent Cells

Culture cells overnight on 22 mm x 22 mm glass coverslips placed in optical grade 6-well tissue culture plates at a density of 4.0 X 10⁵ cells/well. 

1. Pull off culture medium and add 10% formalin to each well. Let sit at room temperature for 10 minutes.

2. Take off the formalin and permeabilize cells as follows, depending on protein type:

a. For detection of either nuclear or mitochondrial protein, add 1 mL TBS with 0.5% Triton X-100.  Incubate for 10 minutes at room temperature. 

b. For detection of a cytoplasmic or membrane protein, add 1 mL PBS/ with 0.5% Tween-20 for 10 minutes at room temperature.

3. Remove permeabilization buffer and add 1 mL PBS 0.1% Tween-20.  not letting the specimen dry out. Wash 3 times for 5 minutes before proceeding to blocking step.

4. To block nonspecific antibody binding, incubate in 1 mL 10% normal serum  from the host of the secondary antibody for 1 hour at room temperature. 

5. Add primary antibody at appropriate dilution (diluted in block: 10% NGS normal serum) and incubate overnight at 4 °C.

6. Remove primary antibody solution and replace with 1 mL PBS. Wash 3 times for  5 minutes in PBS 0.1% Tween-20.

7. Add secondary antibody (diluted in block: 10% normal serum) at appropriate dilution as per manufacturer’s recommendations.  Incubate for 1 hour at room temperature.

8. Remove antibody and replace with PBS, wash  5 minutes in PBS with 0.1% Tween-20.  For second wash, add Hoechst 33258 to PBS 0.1% with Tween-20 at 1:25,000 and incubate for 10 minutes.  Wash a third time, adding phalloidin at 1:40 diluted in PBS with 0.1% Tween-20 for 20 minutes (total of 3 X 5 minute PBS + Tween-20 washes).

9. Carefully remove the coverslips from the wells and blot to remove any excess PBS. Dispense 1 drop of anti-fade mounting medium onto the microscope slide per coverslip. Mount the coverslip with the cells facing towards the microscope slide and tack edges down using clear nail polish.

10. Visualize using a fluorescence microscope and filter sets appropriate for the label used (please see list below). Slides can also be stored in a slide box at < -20 °C for later examination.