IF Protocol for General Cells
GUIDELINE
Immunofluorescence utilizes a specific pairing between antigen and antibody to identify the deposition of abnormal molecules and proteins in the tissue or cell culture sample. Immunofluorescence (IF) staining labels a specific target antigen with a fluorescent dye such as fluorescein isothiocyanate or cyanine. The fluorescent stain visualized under a fluorescent microscope allows for an assessment of the target molecule / protein distribution in the sample. The following protocol provides a method of immunofluorescence of general cells.
METHODS
- A multi-well plate (Glass bottom, 96-well, 300 μL) is coated with fibronectin (12.5 μg/mL) for 1 hour at room temperature (RT).
- Cells are seeded (10,000-15,000 cells per well) and incubated at 37˚C in humidified air with 5.0% CO2 for at least 4 hours.
- Growth medium is removed and the cells are washed in PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.2).
- The cells are fixed for 15 minutes in ice cold 4% paraformaldehyde (pH 7.2-7.3) in growth medium supplemented with 10% fetal bovine serum (FBS).
- The cells are permeabilized 3 times for 5 minutes each with 0.1% Triton X-100 in PBS.
- The cells are washed once with PBS.
- The primary antibody is diluted in PBS supplemented with 4% FBS and incubated overnight at 4˚C.
- The following day, the cells are washed 4 times for 10 minutes each with PBS.
- The secondary antibody is diluted to 1 μg/mL in PBS supplemented with 4% FBS and incubated for 1.5 hours at room temperature.
- The cells are counterstained for 4 minutes with the nuclear probe DAPI.
- The cells are washed 4 times for 10 minutes each with PBS before mounted in PBS containing 78% glycerol.
NOTES
- Cells should be grown, treated, fixed, and stained directly in multi-well plates, chamber slides, or on coverslips.
- All incubations should be carried out at RT unless otherwise noted in a humid light-tight box or covered dish to prevent drying and to prevent exposure of fluorochrome to light.
