Prestige Antibodies® have been used for subcellular localization studies by immunocytochemistry (ICC) - immunofluorescence (IF) using the protocol described below. In addition to the primary antibodies, a set of reference markers are used in each image; an antibody based marker against microtubules as well as the nuclear dye DAPI.
All washes are performed at room temperature.
- A multiwell glass-bottomed plate, suited for cell culturing and high-resolution fluorescent imaging, is coated with fibronectin (concentration 12.5 μg/mL) for 1 h at room temperature.
- Cells are seeded (10,000-25,000 cells per well) and incubated at 37 °C in humified air with 5.0% CO2, for 20-24 h.
- 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 min in 4% paraformaldehyde prepared in PBS.
- The cells are permeabilized for 10 min with 0.1% Triton X-100 in PBS.
- The cells are washed with PBS once and incubated for 1 h at room temperature in blocking buffer (4% FBS in PBS) to block non-specific sites.
- Cells are incubated overnight at 4 °C with primary antibodies in blocking buffer.
- The following day the cells are washed 4x10 min with PBS and incubated for 1.5 h at room temperature with secondary antibodies in blocking buffer.
NOTE: The secondary antibodies are fluorescently labeled and thus light sensitive. The sample should be kept in dim light in this as well as in the following steps.
- The cells are counterstained for 5 min with the nuclear stain DAPI at 1ug/mL in PBS.
- The cells are washed 4x10 min with PBS and mounted in PBS containing 0,02 % (w/v) NaN3.
- Primary antibodies at a working concentration of 1-4 μg/mL.
NOTE: The dilution of the primary antibody is to be considered as a guideline only. Optimal dilution must be determined by the user.
- Mouse anti-alpha Tubulin monoclonal antibody (T9026) diluted 500x for polyclonal antibodies.
- Mouse anti-alpha Tubulin monoclonal antibody, clone DM1A (T9026) diluted 500x for monoclonal antibodies.
Anti-rabbit IgG and anti-mouse IgG fluorescent-dye conjugated secondary antibodies.