Prestige Antibody Immunofluorescence Procedure

A large number of the Prestige Antibodies have been used in subcellular localization studies by immunofluorescence (IF) staining of three cell lines; A-431, U-2 OS and U-251MG. Each cell line is stained with a Prestige Antibody, two organelle probes specific for the endoplasmatic reticulum and micro-tubules, as well as counterstained with the nuclear probe DAPI. The IF images can be viewed on the Human Protein Atlas web portal (www.proteinatlas.org).

Reagents:
Primary antibodies:

• Prestige Antibodies at a working concentration of 1-4 μg/ml.
      Note 1: The dilution of the primary antibody is to be considered as a guideline only.
      Optimal dilution must be determined by the user.
• Chicken anti-Calreticulin polyclonal antibody
• Mouse anti-alpha Tubulin monoclonal antibody

Secondary antibodies:

• anti-mouse IgG -Alexa Fluor^® 555 produced in goat
• anti-chicken IgG -Alexa Fluor 647 produced in goat
• anti-rabbit IgG -Alexa Fluor 488 produced in goat

Protocol:
Cell Seeding
All washes are performed at room temperature (RT).

1. A glass-bottomed, multiwell plate is coated with fibronectin (conc. 12.5 μg/ml) for 1 h at room temperature.
2. Cells are seeded (10,000-15,000 cells per well) and incubated at 37°C in humidified air with 5.2 % CO2, for 4 hours.

Immunostaining

1. Growth medium is removed and the cells are washed in 1x PBS (8.1 mM Na2HPO4, 1.5mM KH₂PO₄, 137 mM NaCl, and 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 3 times for 5 minutes each with 0.1 % TRITON® X-100 in PBS.
4. The cells are washed with 1x PBS and incubated overnight at 4 °C with the primary antibodies in 1x PBS supplemented with 4 % FBS.
5. The following day the cells are washed 4 times for 10 minutes each with 1x PBS and incubated for 1.5 hours at room temperature with the secondary antibodies in 1x PBS supplemented with 4% FBS.
       Note 2: 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 (0.6 μM in 1x PBS).
7. The cells are washed 4 times for 10 minutes with 1x PBS and then mounted in glycerol + 10 % 10x PBS.
   
   
   

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