Double immunofluorescence: sequential protocol
实验概要
We provide a protocol for immunofluoresent double staining incubating the antibodies separately.
实验步骤
1. 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.
2. 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.
3. 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.
