Immunocytochemistry ICC

Summary

Localized detection of antigen expression

Principle

Immunohistochemistry, also known as immunocytochemistry, is a new technique for the qualitative, localization and quantitative determination of the corresponding antigens through antigen-antibody reaction and histochemical chromogenic reaction in tissue cells in situ with specific antibodies labeled with chromogenic agents.

Appliance

Detection of various antigenic substances

Operation method

Immunocytochemistry ICC

Materials and Instruments

【Materials】Sample.
【Reagents, Kits】Cold methanol, acetone, cold PBS, Hoechst or DAPI (DNA stain), BSA, Triton X-100

Move

1. Fixation1) Fix samples in cold methanol, acetone for 1-10 minutes, or PBS with 3-4% paraformaldehyde pH 7.4 for 15 minutes at room temperature.2) Rinse samples twice with cold PBS.2. Penetration1) Incubate samples for 10 minutes in PBS containing 0.25% Triton X-100 (or 100 μM digitalis saponin or 0.5% saponin), the most commonly used detergent to enhance antibody penetration. Triton X-100 is the most commonly used detergent and enhances antibody penetration. It is not suitable for use with membrane-bound antigens because of its damaging effect on cell membranes.2) Rinse cells with PBS for 3 X 5 minutes. 3.3. Blocking and Incubation1) Incubate cells in PBST containing 1% BSA for 30 minutes to block non-specific binding of the antibody (the blocking solution may also contain 1% gelatin or 10% serum from the same animal as the secondary antibody).2) Cells are co-incubated with the antibody (diluted in PBST containing 1% BSA) in a wet box for 1 hour at room temperature or overnight at 4 °C. The cells are then incubated with the antibody (diluted in PBST containing 1% BSA) for 30 minutes to block non-specific binding of the antibody.3) Discard liquid and rinse cells with PBS for 3 x 5 minutes.4) Incubate cells with secondary antibody in 1% BSA for 1 hour at room temperature, protected from light.5) Discard secondary antibody solution and rinse cells with PBS for 3 x 5 minutes, protected from light.4. Re-staining1) Incubate cells with 0.1-1 μg/ml Hoechst or DAPI (DNA stain) for 1 min.2) Rinse with PBS.5. Sealing1) Add a drop of sealing solution to the coverslip to seal it.2) Seal the coverslip with nail polish to prevent it from drying out and transfer to a microscope.3) Store at -20 °C or 4 °C away from light.

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