Confocal microscopy of the uptake of Hx-heme and receptor-binding α₂-macroglobulin in COS-1 cells. (A) Immunostaining of cells incubated 2 minutes at 37°C with Alexa 488-labeled Hx-heme (green color) and monoclonal rhodamine-labeled anti-LRP/CD91 antibody (red color). Notice the colocalization of the 2 fluorochromes. (B) Cells incubated 2 minutes at 37°C with Alexa 488-labeled Hx-heme (green color) and immunostaining for EEA1 using Alexa 594-labeled secondary antibody (red color). (C) Cells incubated for 60 minutes with Alexa 488-labeled Hx-heme (green color) and immunostained with LRP/CD91 using Alexa 594-labeled secondary antibody (red color). Notice the distinct staining of the 2 fluorochromes. (D) Vesicular staining of COS-1 cells incubated for 60 minutes with Alexa 488-labeled Hx-heme (green color) and receptor-binding (activated) α₂-macroglobulin (α₂M^*), which was immunostained by an Alexa 594-labeled secondary antibody (red color). Notice the overlapping coloring in the overlay picture (right panel). Optic magnification was × 630 in all the figure displays.