Immunocytochemistry protocol (ICC protocol)

Immunocytochemistry (ICC) is classically defined as a procedure to detect antigens in cellular contexts using antibodies. Immunocytochemistry (ICC) is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a particular sample express the antigen in question. In cases where an immunopositive signal is found, ICC also allows researchers to determine which sub-cellular compartments are expressing the antigen.

Same to immunofluorescence assay, there are also two different immunocytochemistry (ICC) assay which include indirect immunocytochemistry (ICC) assay and direct immunocytochemistry (ICC) assay. 
In indirect immunocytochemistry (ICC) test, the protocol includes preparation of tissue or cell, tissue or cell fixation, serum blocking, primary antibody incubation, marked second antibody incubation, staining, result judgment and imaging. In direct immunocytochemistry (ICC) test, there are only marked primary antibody been incubated without second antibody and other steps are same.

Indirect Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip 
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each
11. Incubate samplels with fluorochrome-conjugated secondary antibody at appropriate dilution in PBS for 1 hour at 37°C in dark(optimal condition should be confirmed in different l aboratory)
12. Rinse three times in PBS for 5 min each in dark
13. Incubate samples with 1 μg/ml DAPI
14. Mount samples with a drop of mounting medium

Direct Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip 
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with fluorochrome- conjugated primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each in dark
11. Incubate samples with 1 μg/ml DAPI
12. Mount samples with a drop of mounting medium

Protocol of Immunocytochemistry (ICC)?