ICC-IF Standard Protocol
The protocol described below is the Atlas Antibodies standard protocol for subcellular localization studies by immunocytochemistry (ICC) with immunofluorescence (IF) detection. The protocol is optimized for Triple A Polyclonals and PrecisA Monoclonals.
Download a pdf version of the standard protocol for ICC-IF.
In addition to the primary antibodies, a set of reference markers are used in each image displayed in our product catalog: 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 (Greiner bio-one, product no: 655891) 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.
- The 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 the dim light in this as well as in the following steps.
- The cells are counterstained for 5 min with the nuclear stain DAPI (Invitrogen) at 1ug/ml in PBS.
- The cells are washed 4x10 min with PBS and mounted in PBS containing 0,02 % (w/v) NaN3.
The following reagents are used:
- Triple A Polyclonals and PrecisA Monoclonals 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 (Sigma-Aldrich) diluted 500x for Triple A Polyclonals.
- Rabbit anti-alpha Tubulin monoclonal antibody ab52866 (Abcam) diluted 1000x for PrecisA Monoclonals.
The following secondary antibodies are used for the Triple A Polyclonals and PrecisA Monoclonals
- Alexa® Fluor 594 goat anti-mouse IgG (ThermoFisher Scientific, product no: A11032) diluted 400x.
- Alexa® Fluor 488 goat anti-rabbit IgG (ThermoFisher Scientific, product no: A11034) diluted 400x.