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 2nd 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). DonÕt 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