IF PROTOCOL

By admin

Immunofluorescence (IF) staining is a technique widely used in biological research and clinical diagnosis that uses fluorescently labeled antibodies to detect specific target antigens. Staining is very intuitive and results can be observed directly. Although it is a well-established technique, several factors must be considered and various optimization steps must be taken to ensure successful staining.

Experimental steps:

Immunostaining of Cultured Cells

All steps are performed at room temperature unless otherwise stated. For best staining results, unless the cell line used is fragile (e.g., neuronal cells), the procedure should be performed on a slow rotating shaker.

I. Fixed and permeable:

1. Aspirate the medium and wash the cells inoculated onto clean coverslips with 1X PBS.

2. Cells were fixed for 15 minutes with fresh 4% paraformaldehyde in 1X PBS.

3. Rinse the coverslips 3 times for 3 minutes each with 1X PBS.

4. Permeabilize with 0.2% Triton X-100 in 1X PBS for 5 minutes.

5. Rinse the coverslips 3 times for 3 minutes each with 1X PBS.

II. Blocking:

1. Prepare 5% normal serum in 1X PBS as the blocking solution. Select serum from the same species as the secondary antibody, e.g. if the secondary antibody is goat anti-mouse, goat serum should be selected as the blocking solution.

2. Incubate the cells with the containment solution for 1 hour (or if the appropriate serum is not available, use 3% BSA in 1X PBS for containment).

III. Antibody Incubation:

1. Aspirate the blocking solution and dilute the primary antibody in Antibody Diluent. Prepare a blank control (incubate in antibody diluent without primary antibody). Incubate for 1 hour at room temperature or overnight at 4°C.

2. Note: If incubating overnight, ensure that the coverslips do not dry out.

3. Wash the specimen 3 times for 5 minutes each with 1X PBST.

4. Add the appropriate amount of fluorescent secondary antibody diluted with Antibody Dilution Buffer and incubate for 1 hour at room temperature in a humid, dark environment.

5. Note: After adding the fluorescent secondary antibody, the samples must be kept in the dark as long as possible.

6. Wash the specimen 3 times for 5 minutes each time with 1X PBST (0.1% Tween).

IV. Sealing and Microscopy:

1. Seal the specimen on the slide with a sealer containing DAPI (if required).

2. Observe the slide under a fluorescence microscope.