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Double immunofluorescence: sequential protocol

Procedure for immunofluoresent double staining incubating the antibodies separately

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See our fixation and permeabilization protocol.  

Blocking and sequential incubation

  1. First blocking step: incubate cells with the first serum (10% serum from the species that the secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 1% BSA) at room temperature.
  2. Incubate cells with the first primary antibody in 1% BSA or 1% serum in PBST in a humidified chamber for 1 hr at room temperature or overnight at 4°C depending on the concentration of the antibody and the accessibility of the antigen.
  3. Decant the first primary antibody solution and wash the cells three times in PBS, 5 min each wash.
  4. Incubate cells with first secondary antibody (labelled with Fluorochrome-1) in 1% BSA in PBST for 1 hr at room temperature in dark.
  5. Decant the first secondary antibody solution and wash three times with PBS for 5 min each in dark.
  6. Second blocking step: incubate cells with the second serum (10% serum from the species that the secondary antibody was raised in) for 30 min to block unspecific binding of the antibodies (alternative blocking solutions are 1% gelatin or 1% BSA) at room temperature in the dark.
  7. Incubate cells with the second primary antibody in 1% BSA in PBST in a humidified chamber in the dark for 1 hr at room temperature or overnight at 4°C depending on the concentration of the antibody and the accessibility of the antigen.
  8. Decant the second primary antibody solution and wash the cells three times in PBS, 5 min each wash in dark.
  9. Incubate cells with second secondary antibody (labelled with Fluorochrome-2) in 1% BSA for 1 hr at room temperature in dark.
  10. Decant the second 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 in dark.
  2. Rinse with PBS in dark.

Mounting

Mount coverslip with a drop of mounting medium.

  1. Seal coverslip with nail polish to prevent drying and movement under microscope.
  2. Store in dark -20°C or 4°C.

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