Protocols - Immunofluorescence: General Cell
Please refer to the antibody Product Information Sheet for the primary antibody concentration and the selected cell line.
- A multiwell plate (Glass bottom, 96-well, 300μL) is coated with fibronectin (conc. 12.5μg/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 (8.1mM Na2HPO4, 1.5mM KH2PO4, 137mM NaCl, 2.7mM 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 1μg/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.