Basic ICC Protocol – dishes or wells

*Please read “Notes” prior to using this protocol for the first time.

1. Remove culture media and replace with PBS. If cells detach easily or are sensitive to an air interface, remove ¾ of the media and replace with PBS.  Repeat twice more.
2. Replace PBS with 2ml of 2% Fixative and fix for 10min at RT. If cells detach easily or are sensitive to an air interface, remove 1/2 of the final PBS wash and add an equal volume of 4% fixative to give a final conc. of 2%.
3. Rinse wells with PBS 3 X 5min @. (Prepare antibodies in 10% serum from the host of the secondary antibody)
4. Remove PBS one well at a time and add ice-cold 100% Methanol.  Keep samples on ice for 3min to permeabilize.             
5. Rinse wells with PBS 3 X 5min at RT.
6. Remove PBS one well at a time and add prepared primary antibody.  Add 10% serum/PBS to controls.
7. Place the dishes in a dark box and incubate overnight at 4°C.  Slight agitation is good, but not necessary.
8. Rinse wells in PBS for 3x10min with agitation. (Prepare secondary antibodies in 10% serum from the host of the secondary.)
9. Remove PBS one well at a time and add prepared secondary antibody.  Add 10% serum/PBS to controls.
10. Incubate in the dark for 1hr. at RT. Slight agitation is ok, but not necessary.
11. Wash samples in PBS for 3x10min with agitation.  Protect from light.
12. If desired, after final wash, replace PBS with 300nM DAPI, just enough to cover, for 1min. (**DAPI is mutagenic.  Use caution and discard used solution as chemical waste!)
13. Replace DAPI with PBS promptly and agitate for 2min at RT.  Remove and add fresh PBS.
14. Mount coverslips using anti-fade mounting medium. (MOWIOL, ProLong, VectaShield)

NOTES:  This protocol should be viewed as a guide – the steps can be modified according to need!  Please check your cell and antibody datasheets for specific conditions that may influence the steps you include, exclude or alter.

• Fixative concentration can be as high as 4% if the antigen of interest is known to be unaffected by crosslinking.  Time and concentration can be altered as needed.
• Cold acetone can also be used for 1-10min on ice or in the freezer – further permeabilization is not necessary.  
• Permeabilization can also be achieved using 0.1-0.3% Triton/PBS for 10min at room temp.
• Permeabilization is not appropriate for membrane-bound antigens!
• Unpurified primary antibodies can cause background issues.  Add 1% BSA to the diluted primary antibody.  Including a blocking step of 30min in 1%BSA/PBS just prior to primary antibody incubation will also help to reduce non-specific primary antibody binding.
• More rigorous permeabilization can include a 30min blocking/permeabilization step prior to incubation with the primary antibody using 1%BSA/0.1-0.3% Triton/PBS.   Dilute the primary antibody in this solution  +10% serum from the host of the secondary antibody for the overnight incubation.  (Methanol incubation is omitted, in this case!)
• Optimum fixation/permeabilization/blocking conditions should be tested for each cell type and antibody.
• Handle DAPI with care – it has mutagenic properties.  DAPI wastes, including washes, should be collected separately as chemical wastes.