Live cell indirect immunofluorescence

One. experiment equipment:

1. equipment:

40-well plastic plate, 40-well plate centrifuge, micro sampler, oscillator, coverslip, fluorescence microscope, etc.

2. Reagents:

Monoclonal antibody (mAb), fluorescently labeled anti-mouse immunoglobulin, PBS (pH7.2-7.4), glycerin, sodium azide (NaN3), etc.

two. Method (Micro Method)

1. The separated mononuclear cells were washed twice with PBS at 1000 rpm for 10 minutes each. Adjust the cell concentration to 0.5-1 × 108 / ml (50 million-100 million / ml) with PBS containing 0.1% NaN3, add it to a 40-well plate, 10ul per well, containing 5-10 × 105 cells, then add monoclonal antibody (Primary antibody) 50ul (simultaneously add 5ul of AB type serum) and mix by shaking, set at 4 ℃ for at least 30 minutes.

2. Wash once with PBS containing 0.1% NaN3, centrifuge at 1000 rpm for 3-5 minutes and discard the supernatant.

3. Add 20ul of fluorescently labeled anti-mouse antibody (second antibody) working solution, mix and shake for 4 ℃ / 30 minutes (the time cannot exceed).

4. Wash twice with PBS containing 0.1% NaN3. The method is the same as above. Discard the supernatant, add 5-10ul of PBS containing 60% glycerol (depending on the amount of cells added) and oscillate evenly.

If you want to see the results the next day, add 1% paraformaldehyde PBS to each well after the third step, 50ul per well, mix and place at 4 ℃ overnight, centrifuge the supernatant the next day and observe the results in four steps (for membrane fluorescence).

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