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.

Operation method

Immunofluorescence labeling of suspension 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

Suspension Cells
Poly-L-Lysine
Centrifuge Incubator

Move

1. Cells were cooled in an ice bath and then centrifuged in a benchtop centrifuge at 800 g for 5 min at 4°C. The culture medium was aspirated and the cells were resuspended in 4°C PBS.2. Centrifuge and aspirate the PBS, resuspend the cells with 1~2 ml of 2% PFA fixative, or 2% PFA fixative/0.1% Triton X-100, and fix on ice for 30 min.
3. Centrifuge, aspirate the fixative, resuspend the cells with 15 ml of 4℃ PBS, leave for 5 min, and then wash the cells with PBS again.

4. Dilute the primary antibody in a microcentrifuge at 13,500 g for 2 min at 4°C. 250 μl of antibody is sufficient for ^5x106 cells.5. Centrifuge the cells, aspirate the PBS, resuspend the cells in the primary antibody, and incubate at 4°C for 1 h. The cells were incubated for 1 h at 4°C.
6. Dilute cells and antibody to 15 ml with 4°C PBS.7. Centrifuge, aspirate the PBS and resuspend the cells in 4°C PBS and repeat once.8. secondary antibody 4°C centrifuge with microcentrifuge at 13 500 g for 2 min, ^5x106 cells with 250 ul antibody is sufficient.

9. Aspirate off PBS, resuspend cells with secondary antibody and incubate at 4°C for 1 h. Repeat steps 6 and 7.

10. Unless the cells are to be observed immediately, wrap the centrifuge tube in aluminum and refrigerate before proceeding to the next step of cell concentration.11. Centrifuge the cells and resuspend them in a small amount of PBS (not water), place a drop of the cell suspension on a poly-L-lysine-coated slide, add a coverslip and observe the results as soon as possible.

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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