Cellular IF Labeling Protocol
GUIDELINE
Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen, especially by an image analyzer. We provide the process of cells staining.
METHODS
- Cell Preparation. Remove cells from incubator. Inspect under inverted light microscope to verify the desired appearance. Discard medium. Then, rinse with PBS, remove excess solution.
- Fixation. A. Methanol-Acetone Fixation. Fix in cooled methanol, 10 minutes at -20 °C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at -20 °C. B. Paraformaldehyde-Triton Fixation. Fix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 2-10 minutes. C. Paraformaldehyde-Methanol Fixation. Fix in 3-4 % paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at -20 °C. D. PEM-Ethanol Fixation. Fix in PEM buffer for 10 minutes. Rinse twice, briefly, with PBS. Permeabilize with cooled ethanol for 5-10 minutes at -20 °C. then, wash three times (at least 5 minutes each) with PBS.
- Application of Primary Antibody. Dilute primary antibody in PBS to appropriate dilution. Apply on coverslips over the cells and incubate for 60 minutes at room temperature (it is recommended to use a humidified chamber). Then, wash three times (at least 5 minutes each) with PBS.
- Application of Secondary Antibody. Dilute labeled secondary antibody to appropriate dilution in PBS. Apply on coverslips and incubate for 30 minutes at room temperature. Then, wash three times (at least 5 minutes each) with PBS and remove excess PBS.
- Evaluation. Mount coverslips with mounting medium and invert onto glass slides. Inspect under the microscope. Record the results. It is recommended to photograph the labeled cells.
NOTES
- Secondary antibody is applied only in indirect assays.
- It is advisable to run the appropriate negative controls. Negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies. The ideal negative control reagent is a fluorochrome conjugated mouse monoclonal or myeloma protein.
