Immunofluoresence Transferrin-Uptake Assay

 

Incubate cells in serum free medium 1hr-0/n

 

Apply 25 µg/ml biotinylated holo-transferrin (Sigma T-3915, from stock 1 mg/ml) in pre-warmed serum free medium to cells and incubate at 37 °C for the desired time (15-20 minutes for steady state).

 

Rinse briefly with PBS

 

Fixation temperature may range from 4°C to 37°C, depending on other stains. For transferrin alone, use 4°C.

 

Important: Fix cells with 3.2 % paraformaldehyde pre-equilibrated in serum-free medium at the required temperature for 20 min.

 

Rinse with PBS and quench for 10 min with 50 mM NH₄Cl in PBS

 

Permeabilise and block with TBS + 0.2 % Saponin + 10 % serum derived from the animal species in which the 2^nd antibody was raised for 15 min.

 

Use 1:10 dilution of above dilution in TBS (0.02 % Saponin + 1 % serum) to make a 1:500 dilution of Streptavidin from Molecular Probes conjugated to the fluorophor of choice (e.g. Alexa 546). Don’t use more than 1:500. Incubate cells for 0.5 -1 hour or o/n at 4°C.

 

Wash three times in TBS + 0.02 % Saponin + 1 % serum

 

Dip rinse in distilled water.

 

Mount in a good anti-fade