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:
   1. 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.
   2. 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.
     

    Fluorescent Dyes	λabs (nm)	λfl (nm)
    Atto390	390	479
    Atto425	436	484
    Atto465	453	508
    Atto488	501	523
    Atto532	532	553
    Atto565	563	592
    Atto590	600	627
    Atto594	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