Introduction to Immunofluorescence Technology

Immunofluorescence technique (also known as fluorescent antibody technology), according to the principle of antigen-antibody reaction, fluorescein is coupled to a known antigen or antibody to make a fluorescent probe, which exists in serum or body fluids, cells and tissues. Corresponding antibodies (or antigens) bind to detect qualitative, quantitative and/or localization of antigens or antibodies present in these tissues.

For fluorescently labeled antibodies, the following principles should be followed: specificity, high purity, and satisfactory titer (>1:20), preferably using monoclonal antibodies.

Fluorescein for antibody labeling requires the following conditions: it can form stable covalent bonds with proteins; it does not affect the biological activity of proteins and itself after labeling; high fluorescence efficiency, no significant decrease in fluorescence intensity after labeling; fluorescent color and background The color contrast is clear; the marking method is simple and fast; safe and non-toxic.

The principle of preservation of fluorescent antibodies is to prevent antibody inactivation; to keep fluorescein not falling off and not quenching.

The staining methods of immunofluorescence techniques are divided into direct and indirect methods. The principle of the direct method is to directly react the fluorescently labeled antigen/antibody with the corresponding antibody/antigen, and is mostly used for flow cytometry analysis. The advantage of the direct method is that it is easy to operate, has high specificity, and has few non-specific fluorescent dyes. However, the sensitivity of the direct method is low, and a fluorescent antibody needs to be prepared for each antigen to be detected. If a plurality of antigens are detected, a plurality of corresponding fluorescently labeled antibodies are prepared. The principle of the indirect method is to first label the unlabeled antibody, bind the antigen antibody sufficiently, and then wash to remove the unbound antibody; then add the fluorescently labeled secondary antibody to bind to the primary antibody that has been bound to the antigen. .