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Precautions for the selection of immunofluorescence double-labeled antibodies

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Double immunofluorescence

When detecting two antigens at the same time in the same tissue or cell sample, double fluorescent staining is required. Double immunofluorescence labeling method (double immunofluorescence labeling method) is also divided into direct method and indirect method.

Direct double immunofluorescence labeling

Direct double immunofluorescence labeling: mix two different fluorescein-labeled antibodies (such as anti-A and anti-B) in an appropriate ratio, drop them on the specimen and incubate, then wash off the unbound fluorescent antibody, under a fluorescence microscope Select two corresponding excitation filters for observation, and then locate and quantify the two antigens. The direct method is simple and reliable, but the sensitivity is low.

Indirect double immunofluorescence labeling

Indirect double immunofluorescence labeling: incubate tissues or cells with two unlabeled specific primary antibodies (from different species), wash off the excess primary antibodies, and then incubate with two different fluorescein-labeled secondary antibodies The tissues or cells are washed away with excess secondary antibodies, and then two corresponding excitation filters are selected for observation under a fluorescence microscope, so as to locate and quantify the two antigens.

immunofluorescence labeling method


  1. The primary antibodies are of different species, preferably monoclonal antibodies, the secondary antibodies match the corresponding primary antibodies, and the secondary antibodies have different fluorescent labels. If possible, the secondary antibodies are preferably from the same host species.

  2. When the primary antibody is a monoclonal antibody: the secondary antibody should target the class or subclass of the primary antibody. For example, a mouse IgM primary antibody requires an anti-mouse IgM secondary antibody. When using multiple subclass-specific primary antibodies, subclass-specific secondary antibodies should be used to distinguish the primary antibodies. For example, double-labeled immunofluorescence experiments using IgG2 and IgG1 primary antibodies are better to use anti-IgG2 and anti-IgG1 secondary antibodies.

  3. When the primary antibody is a polyclonal antibody: IgG secondary antibody can be used, because most polyclonal antibodies are IgG immunoglobulins.

  4. In order to limit the non-specific binding of the secondary antibody to the primary antibody of the closely related species, use the secondary antibody pre-adsorbed to the closely related species. For example, the anti-mouse secondary antibody may cross-react with the rat primary antibody. The use of anti-mouse secondary antibodies adsorbed by rat serum can reduce cross-reactivity. Note: Carefully select secondary antibodies that are pre-adsorbed to closely related species (such as rat and mouse). Adsorption can eliminate high-affinity antibodies, leading to weaker signals.

  5. Selection of blocking solution: The function of blocking solution is to try to block off antigens that can bind to the secondary antibody to produce non-specific staining. Therefore, try to choose a serum blocking solution that is homologous to the secondary antibody species. For example, if the secondary antibody is “donkey anti-goat AF488, donkey anti-rabbit AF594”, then the best blocking solution is donkey serum blocking solution. The best situation is that in the immunofluorescence double-labeling experiment, the species of the secondary antibodies are derived from non-mouse, non-rabbit, and non-goat, but donkey, horse, chicken and other species are selected, and the secondary antibodies use the same species source. In this way, one source of serum can complete the blocking of two antibodies.

  6. Incubate the two primary antibodies simultaneously, and then incubate the two secondary antibodies simultaneously. The antibody concentration and incubation time should be explored by yourself. It is better to incubate the primary antibody overnight at 4°C.

  7. Observation is generally completed within 1 hour after fluorescent staining, or stored at 4°C for 4 hours. If the time is too long, the fluorescence may decline prematurely.

Case 1

Sample species: rat
Primary antibody: A (mouse anti-rat), B (rabbit anti-rat)
Secondary antibody: A (goat anti-mouse), B (goat anti-rabbit)

This is the most common and the easiest. The blocking solution selects goat serum for the secondary antibody. Generally use 10% normal goat serum 30 min 37℃.

Case 2

Sample species: rat
Primary antibody: A (mouse anti-rat), B (rabbit anti-rat)

Secondary antibody: A (goat anti-mouse), B (donkey anti-rabbit)
The blocking serum at this time is BSA.

Related Articles:

Fluorescent Antibody Technique (Direct, Indirect)

Fluorescent Antibody Technique

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