The cells were fixed and primary and secondary antibodies were added sequentially and visualized by UV light.

Operation method

Programme 16.11 Indirect immunofluorescence

Principle

The cells were fixed and primary and secondary antibodies were added sequentially and visualized by UV light.

Materials and Instruments

Cells cultured on coverslips Secondary antibody to produce a primary antibody of the species Porcine serum or other sealer D-PBSA
Freshly prepared fixative Use culture medium containing 10% FBS Sealer

Move

1. Wash the coverslips with D-PBSA and place them in a suitable dish. For example, 13 mm coverslips can be used in 24-well plates.

2. Place the dish at -20°C for 10 min, add cold fixative (5 % ethanol acetate solution (at -20°C)) and leave for 20 min.

3. Remove the fixative, wash the coverslips in D-PBSA, add 1 ml of normal porcine serum and leave for 20 min at room temperature.

4. Drench the coverslip in D-PBSA, absorb it with absorbent paper, turn the coverslip over and place it on a drop of 50 μl of diluted primary antibody (diluted 1:100~1:1000 with 10 % FBS culture medium).

5. After 30 min at 37°C, incubate at room temperature for 1-3 hours or overnight at 4°C. If incubated at 4°C, the antibody can be diluted to 1:1000.

6. Drench coverslips in D-PBSA and transfer to 1:20 dilution of secondary antibody (corresponding to a different primary antibody-producing species (e.g., if the primary antibody is produced by a rabbit, the secondary antibody should be from a different species, e.g., goat-rabbit immunoglobulin); the secondary antibody should be labeled with fluorescein or rhodamine) at 37°C for 20 min.

7. Drench the coverslips in D-PBSA and seal the slides with D-PBSA containing 50 % glycerol and fluorescence quenching retardant (Vecta).

8. Observe the slides with a fluorescence microscope.

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