Sequential Double IF Protocol

GUIDELINE

Double immunofluorescence is used to detect the presence and localization of two different proteins or antigens in a biological sample. It involves the use of two different fluorescently labeled antibodies, each targeting a specific protein or antigen of interest. By combining the two antibodies, researchers can visualize and study the distribution and co-localization of the two proteins in a cell, tissue, or organism. This technique is commonly used in research areas such as immunology, cell biology, and pathology. We provide a protocol for immunofluorescent double staining incubating the antibodies separately.

METHODS

Blocking and sequential incubation

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.
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.
Decant the first primary antibody solution and wash the cells three times in PBS, 5 min each wash.
Incubate cells with first secondary antibody (labelled with Fluorochrome-1) in 1% BSA in PBST for 1 hr at room temperature in dark.
Decant the first secondary antibody solution and wash three times with PBS for 5 min each in dark.
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.
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.
Decant the second primary antibody solution and wash the cells three times in PBS, 5 min each wash in dark.
Incubate cells with second secondary antibody (labelled with Fluorochrome-2) in 1% BSA for 1 hr at room temperature in dark.
Decant the second secondary antibody solution and wash three times with PBS for 5 min each in dark.

Counter Staining

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

Mounting

Mount coverslip with a drop of mounting medium.
Seal coverslip with nail polish to prevent drying and movement under microscope.
Store in dark at -20°C or 4°C.

NOTES

Choose two primary antibodies with different IgG isotypes. Either different subgroups from the same species or different species. Make sure you have the matching secondary fluorescence antibodies.
Procedure is done at room temperature except for microwaving steps.