Basic ICC Protocol – dishes or wells
*Please read “Notes” prior to using this protocol for the first time.
- 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.
- 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%.
- Rinse wells with PBS 3 X 5min @. (Prepare antibodies in 10% serum from the host of the secondary antibody)
- Remove PBS one well at a time and add ice-cold 100% Methanol. Keep samples on ice for 3min to permeabilize.
- Rinse wells with PBS 3 X 5min at RT.
- Remove PBS one well at a time and add prepared primary antibody. Add 10% serum/PBS to controls.
- Place the dishes in a dark box and incubate overnight at 4°C. Slight agitation is good, but not necessary.
- Rinse wells in PBS for 3x10min with agitation. (Prepare secondary antibodies in 10% serum from the host of the secondary.)
- Remove PBS one well at a time and add prepared secondary antibody. Add 10% serum/PBS to controls.
- Incubate in the dark for 1hr. at RT. Slight agitation is ok, but not necessary.
- Wash samples in PBS for 3x10min with agitation. Protect from light.
- 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!)
- Replace DAPI with PBS promptly and agitate for 2min at RT. Remove and add fresh PBS.
- 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.