Double staining method immunofluorescence histochemical staining steps

If there are two antigens A and B that need to be displayed at the same time in the same specimen, the antibody of A antigen is labeled with FITC, and the antibody of B antigen is labeled with TRITC, and the following staining methods can be used:

Immunofluorescence cytochemical double staining

Primary cultured rat telencephalon cells at day 14 embryos: Under the induction of osteogenesis morphoprotein-, acetylcholine transferase (green fluorescence) and homeodomain protein Islet-1 (red fluorescence) coexist in the cytoplasm (laser scanning coexistence). Focusing microscope observation)

1. In the one-step double staining method, the two fluorescently labeled antibodies were mixed in an appropriate ratio (A+B), and the staining was carried out according to the direct method.

2. Two-step double-staining method is firstly used for immunofluorescence staining with TRITC-labeled antibody A, and then with FITC-labeled antibody B. According to the animal species of the two antibodies, direct or indirect methods can be used. The result is that the A antigen is orange-red fluorescence, while the B antigen exhibits yellow-green fluorescence. In the application, the commonly used method is the combination of the indirect method in immunofluorescence histochemistry and the fluorescent dye SABC-Cy3 method. For example, in the experimental design, mouse anti-rat A and rabbit anti-rat B were selected as two different specific primary antibodies, and the matched secondary antibodies were FITC-anti-mouse IgG and biotinylated-anti-rabbit, respectively. IgG.

When performing double staining, the primary antibodies can be mixed due to the different sources of the two primary antibodies. After the incubation, wash the unbound primary antibody. When adding the secondary antibody dropwise, first add human biotinylated-anti-rabbit IgG (secondary antibody) to incubate. After washing, drop the SABC-Cy3 complex. Specific fluorescence, then incubated with FITC-anti-mouse IgG (secondary antibody), and finally mounted for observation. As a result, the A antigen exhibited green fluorescence, and the B antigen exhibited red fluorescence.

In this method, it should be noted that when the two primary antibodies are mixed and diluted, the final concentration of the antibody should be the working concentration of each antibody. In other words, the mixed liquid should meet the working concentrations of the two primary antibodies at the same time. If the two primary antibodies are from the same species, double staining must be performed twice, that is, the A antigen is first stained with the first primary antibody and the first fluorescent secondary antibody, and then washed with the second primary antibody and the first fluorescent secondary antibody. Two fluorescent secondary antibodies are used to stain B antigen, otherwise cross-reaction will occur and the staining will be non-specific.