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 10^5 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. Take off 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 degrees Celsius.

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.

Fluorescent Dyes            λabs (nm)    λfl (nm)

 Atto390                                   390             479
 Atto425                                   436             484
A tto465                                   453             508
 Atto488                                   501             523
 Atto532                                   532             553
 Atto565                                   563             592
A tto590                                   600             627
A tto594                                   601             627
 Atto633                                   629             657
 Atto637                                   630             659
 Atto655                                   663             684
 Atto680                                   670             710
Atto700                                   700             725
DyLight 488                            493             518
FluoProbes®547H                  557              572
FluoProbes®647H                  653              675
FluoProbes®682                     690              709
FluoProbes®752                     748              772
AMCA                                      346              442
FITC                                        485              535
Rhodamine                             544              576
Texas Red                               595              615