Fig. 3. (A) Raji-DCSIGN cells visualized by total internal reflectance (TIR) illumination of the ventral membrane after fixation and staining for DC-SIGN (DCN46 mAb). (B) Control staining of fixed Raji-DCSIGN cells with the lipophilic dye, DiI under TIR illumination. (C) Addition of 580 kDa FITC-dextran to the medium to demonstrate accessibility of the ventral membrane surface of adhered Raji-DCSIGN cells. Data shown are mean intensities of ventral membrane regions from TIR images before and after addition of the reagent. Ventral ROIs were defined as the largest box that could fit within the cell outline as determined from a DIC image. (D) Control stainings of fixed Raji-DCSIGN cells for membrane-associated proteins, CD46 and CD59. These images were collected in TIRF mode to show only material within 150 nm of the glass substrate. (E) A fixed MDDC stained for DC-SIGN (DCN46 mAb) and visualized by TIR microscopy. (F) 3D projection images to show the dorsolateral membrane distribution of DC-SIGN on fixed Raji-DCSIGN cells as obtained from a deconvoluted z-stack (step=200 nm) of epifluorescence images. (G) DC-SIGN imaged on the dorsal lamellar membrane of a fixed MDDC in a single epifluorescence image focused 2 µm above the coverslip with edge-region detail expanded as indicated. (H) A fixed MDDC imaged for morphology in DIC and for DC-SIGN distribution by deconvolution of a z-stack of epifluorescence images (step=200 nm). Arrows denote active leading edge membrane regions enriched in DC-SIGN. 3D projections are shown as views from 90° (xy plane) and 0° (from the left side of the cell, yz plane). Scale bars, 10 µm.