Internalization pathways of virus in mature and immature MDDCs. Immature (A,C) and mature (B) MDDCs were incubated with AT-2 HIV-1_ADA (3 μg p24/10⁶ DCs, 1 hour at 37°C) and free virus was washed away. The cells were gently resuspended, and approximately 2.5 × 10⁴ cells adhered to alcian blue–treated slides immediately (0 h) or after an additional 2-hour incubation (2 h). Cells were fixed in 4% paraformaldehyde and incubated with biotinylated CD4-IgG₂ (Progenics Pharmaceutical) in addition to murine monoclonals specific for MR, DC-SIGN, EEA1, and LAMP-2 at room temperature in the presence of saponin. After washing, bound biotinylated CD4-IgG₂ was detected with streptavidin-horseradish peroxidase (HRP) and HRP located using the FITC-TSA amplification kit (green).²²  Murine monoclonals were detected with goat antimouse Alexa Fluor 594 (red) and nuclei were stained with DAPI (blue). Overlays of the green and red stains are shown in panels A and B. Separate stains for envelope (env) and LAMP-2 on immature DCs (0 h as an example) are shown in panel C to highlight the limited colocalization of these stains. Original magnification × 100. Double staining for LAMP-2 or MR and envelope was reproduced using 8 different donors, and DC-SIGN and EEA1 repeated with 5 donors. Comparable results were obtained using AT-2 SIV CP-mac–loaded DCs from 2 additional donors (not shown). Murine Ig isotype controls showed no background staining with goat antimouse Alexa Fluor 594 (not shown).