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Please refer to the antibody Product Information Sheet for the primary antibody concentration and the selected cell line.

1. A multiwell plate (Glass bottom, 96-well, 300μL) is coated with fibronectin (conc. 12.5μg/mL) for 1 hour at room temperature (RT).
   
   
2. 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.
   
   
3. 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).
   
   
4. 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).
   
   
5. The cells are permeabilized 3 times for 5 minutes each with 0.1% Triton X-100 in PBS.
   
   
6.  The cells are washed once with PBS.
   
   
7.  The primary antibody is diluted in PBS supplemented with 4% FBS and incubated overnight at 4˚C.
   
   
8. The following day, the cells are washed 4 times for 10 minutes each with PBS.
   
   
9. The secondary antibody is diluted to 1μg/mL in PBS supplemented with 4% FBS and incubated for 1.5 hours at room temperature.
   
   
10. The cells are counterstained for 4 minutes with the nuclear probe DAPI.
    
    
11. The cells are washed 4 times for 10 minutes each with PBS before mounted in PBS containing 78% glycerol.