Prestige Antibody^® Immunofluorescence Procedure

ICC-IF Protocol

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.

Sample Preparation

All washes are performed at room temperature.

1. 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.
2. 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.

Immunostaining

1. 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).
2. The cells are fixed for 15 min in 4% paraformaldehyde prepared in PBS.
3. The cells are permeabilized for 10 min with 0.1% Triton X-100 in PBS.
4. 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.
5. Cells are incubated overnight at 4 °C with primary antibodies in blocking buffer.
6. 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.
7. The cells are counterstained for 5 min with the nuclear stain DAPI at 1ug/mL in PBS.
8. The cells are washed 4x10 min with PBS and mounted in PBS containing 0,02 % (w/v) NaN₃.

Primary Antibodies:

• 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.

Secondary Antibodies:

Anti-rabbit IgG and anti-mouse IgG fluorescent-dye conjugated secondary antibodies.