Immunocytochemistry...

实验概要

The method provides a guideline procedure for staining of cell cultures using immunofluoresence.

实验步骤

1. General procedure

    1) Coat coverslips with polyethylineimine or poly-L-lysine for 1 hr at room temperature.

    2) Rinse coverslips well with sterile H₂O (3 times 5 min each).

    3) Allow coverslips to dry completely and sterilize them under UV light for at least 4 hrs.

    4) Grow cells on glass coverslips or prepare cytospin or smear preparation.

    5) Rinse briefly in phosphate-buffered saline (PBS).

2. Fixation

    1) Fix the samples either in ice-cold methanol, acetone (1-10 min) or in 3-4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature.

    2) Wash the samples twice with ice cold PBS.

Permeabilization:
If the target protein is expressed intracellularly, it is very important to permeabilize the cells. Note: acetone fixed samples do not require permeabilization.

    3) Incubate the samples for 10 min with PBS containing 0.25% Triton X-100 (or 100 μM digitonin or 0.5% saponin). Triton X-100 is the most popular detergent for improving the penetration of the antibody. However, it is not appropriate for the use of membrane-associated antigens since it destroys membranes.

    4) Wash cells in PBS three times for 5 min.

3. Blocking and incubation

    1) Incubate cells with 1% BSA in PBST for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 10% serum from the species that the secondary antibody was raised in).

    2) Incubate cells in the diluted antibody in 1% BSA in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C.

    3) Decant the solution and wash the cells three times in PBS, 5 min each wash.

    4) Incubate cells with the secondary antibody in 1% BSA for 1 hr at room temperature in dark.

    5) Decant the secondary antibody solution and wash three times with PBS for 5 min each in dark.

5. Counter staining

    1) Incubate cells on 0.1-1 μg/ml Hoechst or DAPI (DNA stain) for 1 min.

    2) Rinse with PBS.

6. Mounting

    1) Mount coverslip with a drop of mounting medium.

    2) Seal coverslip with nail polish to prevent drying and movement under microscope.

    3) Store in dark at -20°C or  4°C.