ICC/IF Immunocytochemistry and immuofluorescence protocol

Guideline procedure for staining of cell cultures using immunofluoresence.

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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).

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.

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.

Counter staining

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

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.
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