Immunofluorescence protocol (IF protocol)

Immunofluorescence is one of the widely used techniques in modern biology and medicine, and it is developed by Coons et al. (1950), and it is a combination of immunofluorescence technique and morphological technology to develop immune fluorescent cells (or tissue). The development of fluorescence immunoassay technology is very fast in recent years, especially in the field of medical, biological and environmental studies, it is widely used in the determination of endocrine hormones, growth factors, proteins, nucleic acids, neurotransmitters, receptors, in vivo drug and infectious sources, and so on, these subjects have been developed. According to the detection of different samples can be divided into three major categories.
There are two different immunofluorescence assay which include indirect immunofluorescence assay and direct immunofluorescence assay.For indirect immunofluorescence assay, the protocol mainly include tissue or cell preparation, tissue or cell fixation, serum blocking, primary antibody incubation, marked second antibody incubation, staining, result judgment and imaging. For direct immunofluorescence assay, there are only marked primary antibody been incubated without second antibody and other steps are same.
For direct immunofluorescence assay, specific fluorescent antibody was prepared by the combination of specific antibodies and fluorescein. It is the most simple and fast method for the examination of cell or tissue antigen. This method is highly specific and is commonly used in renal biopsy and pathogen examination. Its disadvantage is that a fluorescent antibody can only examine one kind of antigen, which is less sensitive. For indirect immunofluorescence assay, specific antibodies against the corresponding antigen, fluorescein labeled anti - antibody (anti - specific IgG fluorescent antibody) and the primary antibody
Although the basic steps and principles of immune fluorescence are the same, but because of the specific conditions are not the same, the detailed operation steps of each laboratory will not be exactly the same. For example, the use of the solution, the fixed liquid and antibody dilution liquid will be slightly different. Here to give a more common method, the detailed use of the operation can be based on the steps to adjust and change, and ultimately determine the most appropriate method for youself.

Indirect Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each
11. Incubate samplels with fluorochrome-conjugated secondary antibody at appropriate dilution in PBS for 1 hour at 37°C in dark(optimal condition should be confirmed in different l aboratory)
12. Rinse three times in PBS for 5 min each in dark
13. Incubate samples with 1 μg/ml DAPI
14. Mount samples with a drop of mounting medium

Direct Immunofluorescence / IF

1. Prepare tissue or culture cells
2. Prepare tissue section or cells coverlip
3. Wash samples two times with PBS
4. Fix amples with 4% paraformaldehye in PBS for 15 min at room temperature(Note: Paraformaldehye is toxic, use only in fume hood)
5. Aspirate fixative, rinse two times in PBS for 5 min each
6. Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min(Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.
7. Aspirate triton x-100, rinse two times in PBS for 5 min each
8. Incubate samplels in 10% normal goat serum in PBS for 30 min at room temperature
9. Aspirate goat serum, incubate sections with fluorochrome- conjugated primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C(optimal condition should be confirmed in different laboratory)
10. Rinse three times in PBS for 5 min each in dark
11. Incubate samples with 1 μg/ml DAPI
12. Mount samples with a drop of mounting medium

Protocol of Immunofluorescence (IF)