Fig. 3.

CTB and transferrin receptors do not colocalize. (A) Chilled TF1 cells were simultaneously bound with CTB-488 (green) and anti-transferrin receptor antibodies on ice. Cells were moved to 37°C for dual uptake of both markers for 60 min. After fixation and permeabilization, internalized transferrin receptor antibodies were labeled with Alexa Fluor 594 (red) and visualized by deconvolution microscopy. Note how both biological markers stain separately. Nuclei are counterstained with DAPI (blue). (B) IL-5 binding does not interfere with antibody staining of IL-5 receptors. Chilled TF1 cells were incubated with (+IL-5, hatched line) or without 10 ng/ml IL-5 (–IL-5, solid black line) for 30 min on ice. Cell surface receptors were labeled with the same anti-βc and anti-IL-5Rα-PE-conjugated antibodies used in Figs. 1 and 2, and analyzed by flow cytometry. The shaded histogram represents cells labeled with an isotype-matched control antibody. Note the overlap of –IL-5 and +IL-5 histograms, indicating that the ligand does not interfere with antibody staining.