Immunofluorescent labeling is a commonly used immunological experimental method in microbiology, immunology, pathology and immunohistochemistry.

Operation method

Immunofluorescence labeling assay of monolayer growth cells

Principle

Immunofluorescence technique is to label a known antibody or antigen molecule with fluorescein, and when it reacts with its corresponding antigen or antibody, a certain amount of fluorescein will be carried on the complex formed, and the antigen-antibody binding site emitting fluorescence can be seen under the fluorescence microscope, so that the antigen or antibody can be detected.

Materials and Instruments

monolayer cells
PBS paraformaldehyde methanol antibodies
Centrifuge Water Bath Incubator

Move

1. Place the monolayer of cells grown into sheets on ice to cool, aspirate the culture medium and wash with 4°C PBS. Aspirate off the PBS.

2. For cell surface antigens, fix with 2% PFA on ice for 30 min. For cytoplasmic antigens, fix with 2% PFA containing 0.1% Triton X-100 on ice for 30 min. or with 100% methanol in a freezer (-10~-20°C) for 15 min.3. Aspirate off the fixative and wash twice with PBS at 4°C (5 min each time). The diluted primary antibody is centrifuged at 13,500 g for 2 min at 4°C in a microcentrifuge.4. Add the primary antibody to the petri dish so that it just covers the cells, incubate at 4°C for 1 h, and then wash with 4°C PBS 4 times (5 min each time).5. The diluted secondary antibody is centrifuged at 13,500 g for 2 min at 4°C in a microcentrifuge.6. Add the secondary antibody to the cells and incubate at 4°C for 4 h. Then wash 4 times with 4°C PBS.7. If you do not want to observe the cells immediately, store the cells in PBS. It is important to cover the dish, wrap it in aluminum foil, refrigerate it, and observe the results of this experiment within 24 h because the fluorescence will rapidly diminish or dissociate from the cells.

Caveat

1、Fluorescence staining is usually observed within 1h, or stored at 4℃ for 4h, too long a time may cause fluorescence to decline prematurely.

2、The following three controls should be set for each test：

（1） Positive control: Positive serum + fluorescent marker; (2) Negative control: Positive serum + fluorescent marker.(2) Negative control: negative serum + fluorescent marker.(3) Fluorescent marker control: PBS + fluorescent marker.

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