Immunocytochemistry (ICC) Protocol
GUIDELINE
Immunocytochemistry is a new technique for the qualitative, localized and quantitative determination of the corresponding antigens through antigen-antibody reactions and histochemical colorimetric reactions in tissue cells in situ with specific antibodies labeled with chromogenic agents. It skillfully combines the specificity of immune reaction and the visibility of histochemistry, and detects various antigenic substances (such as proteins, peptides, enzymes, hormones, pathogens and receptors) at the cellular and subcellular levels with the help of the visualization and magnification of microscopes (including fluorescent microscopes and electron microscopes).
METHODS
- Fixation. Fix samples in cold methanol, acetone for 1-10 minutes or PBS containing 3-4% paraformaldehyde pH 7.4 for 15 minutes at room temperature. Then, rinse samples twice with cold PBS.
- Permeability. Incubate samples in PBS containing 0.25% Triton X-100 (or 100 μM digitalis saponin or 0.5% saponin) for 10 minutes. Rinse cells with PBS for 3 × 5 minutes.
- Blocking and incubation. Cells are incubated in PBST with 1% BSA for 30 minutes to block non-specific binding of the antibody. Cells are co-incubated with antibody (diluted in 1% BSA in PBST) in a wet box for 1 hour at room temperature, or overnight at 4°C. Discard the liquid and rinse the cells with PBS for 3 × 5 minutes. Cells are incubated with secondary antibody in 1% BSA for 1 hour at room temperature, protected from light. Discard secondary antibody solution and rinse cells with PBS for 3 × 5 minutes, protected from light.
- Re-stain. 0.1-1 μg/ml Hoechst or DAPI (DNA stain) incubate cells for 1 min. then, rinse with PBS.
- Sealing. Add a drop of sealing solution to the coverslip to seal it. Seal the coverslip with nail polish to prevent it from drying out and move it to the microscope for observation. Store at -20 °C or 4 °C away from light.
NOTES
- Pre-incubation of the sample with 5% BSA for 10 min. prior to the primary antibody reaction may decrease background staining. For best results with animal tissues, use 5 to 10% normal serum from the same species as the host of the secondary antibody.
- Optimal dilution and incubation times should be determined for each primary antibody prior to use.
- When using AEC substrate, do not use alcohol-containing solutions for counter-staining (e.g., Harris' hematoxylin, acid alcohol), since the AEC stain formed by this method is soluble in organic solvents. The slide must not be dehydrated, brought back to toluene (or xylene), or mounted in toluene-containing mountants.
