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Prestige Antibody® Immunofluorescence Procedure

Prestige Polyclonals and Monoclonals have been used for subcellular localization studies by immunocytochemistry (ICC) - immunofluorescence (IF) staining in three to five human cell lines using the protocol described below. Each cell line is stained with a primary antibody, two organelle probes specific for the endoplasmatic reticulum and micro-tubules, as well as counterstained with the nuclear probe DAPI. The ICC-IF images can be viewed on the Human Protein Atlas web portal (proteinatlas.org).

Sample Preparation

All washes are performed at room temperature.

  1. A multiwell, glass-bottomed, plate (Whatman, Maidstone, UK) is coated with fibronectin (concentration 12.5 μg/ml) for 1 hour at room temperature.
  2. Cells are seeded (10.000-15.000 cells per well) and incubated at 37°C in humified air with 5.2% CO2, for at least 4 hours.

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 minutes in ice cold 4% paraformaldehyde pH 7.2 - 7.3 in growth medium supplemented with 10% fetal bovine serum (FBS).
  3. The cells are permeabilized 3x5 minutes with 0.1% Triton X-100 in PBS.
  4. The cells are washed with PBS and incubated overnight at 4°C with primary antibodies in PBS supplemented with 4% FBS.
    NOTE: The specified working dilutions of the primary antibodies are to be considered as a guideline only. Optimal dilutions must be determined by the user.
  5. The following day the cells are washed 4x10 minutes with PBS and incubated for 1.5 hours in room temperature with secondary antibodies in PBS supplemented with 4% FBS.
    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.
  6. The cells are counterstained for 4 minutes with the nuclear stain DAPI (Invitrogen) 0.6 μM in PBS.
  7. The cells are washed 4x10 minutes with PBS and mounted in glycerol + 10% 10xPBS.  

Revision B, Sept 2014.

 

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