Immunofluorescence (IF) Protocol
GUIDELINE
Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualize the location of the antibodies. For direct immunofluorescence assay, there are only marked primary antibody been incubated without second antibody and other steps are same.
METHODS
- Prepare tissue or culture cells.
- Prepare tissue section or cells cover lip.
- Wash samples two times with PBS.
- Fix samples with 4% paraformaldehye in PBS for 15 min at room temperature.
- Aspirate fixative, rinse two times in PBS for 5 min each.
- Permeabilize samples with 0.1-0.5% triton x-100 in PBS for 10 min.
- Aspirate triton x-100, rinse two times in PBS for 5 min each.
- Incubate samples in 10% normal goat serum in PBS for 30 min at room temperature.
- 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.
- Rinse three times in PBS for 5 min each in dark.
- Incubate samples with 1 μg/ml DAPI.
- Mount samples with a drop of mounting medium.
NOTES
- Paraformaldehye is toxic, use only in fume hood.
- 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.
- Optimal condition should be confirmed in different laboratory.
