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.

 

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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.

Sample preparation

All washes are performed at room temperature.

  1. 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.
  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. 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).
  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 the dim light in this as well as in the following steps.
  7. The cells are counterstained for 5 min with the nuclear stain DAPI (Invitrogen) at 1ug/ml in PBS.
  8. The cells are washed 4x10 min with PBS and mounted in PBS containing 0,02 % (w/v) NaN3.

Reagents

The following reagents are used:

Primary antibodies:

  • 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.

Secondary antibodies

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.