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.
