Anti tfr

RPE cells were incubated with 20 μg/ml of A647-Tfn for indicated times at 37°C and then immediately placed on ice and washed three times in ice-cold PBS²⁺ to remove unbound ligand, fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X-100, stained with either anti-EEA1 (Cell Signaling Technology) or anti-TfR (Santa Cruz Biotechnology) and appropriate secondary antibodies, and then mounted in fluorescence mounting me­dium (Dako). Colocalization of A647-Tfn with either EEA1 or TfR was performed in ImageJ using Pearson’s r. The results were subjected to two-way analysis of variance (ANOVA) followed by Bonferonni’s multiple comparison posttest.

DMEM/F12, fetal bovine serum (FBS), penicillin/streptomycin solution, insulin-transferrin-selenium-ethanolamine solution, and sterile 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer were obtained from Life Technologies (Carlsbad, CA), as was Biotin-xx-Tfn. Avidin and o-phenylenediamine hydrochloride reagent were obtained from Biobasic (Markham, Canada), and biocytin was obtained from Santa Cruz Biotechnology (Dallas, TX). Superblock blocking buffer was obtained from Thermo Fisher (Rockford, IL). Antibodies and fluorescent ligands used were as follows: anti-LYCAT from Genetex (Irvine, CA); anti-EEA1 and actin from Cell Signaling Technology (Danvers, MA); anti-TfR from Santa Cruz Biotechnology; Tfn antibodies (used in Tfn uptake assay) from Bethyl Laboratories (Montgomery, TX); and Alexa 647–conjugated Tfn (A647-Tfn) from Life Technologies.