Figure 1: Localization of FRα in human choroid plexus and in FRα-transfected rat choroid plexus Z310 cells.

(a) Immunohistochemical staining of FRα in human choroid plexus specimen shows a predominant localization at the apical brush border membrane. A minor portion of the receptor is localized at the basolateral membrane and an intracellular punctuate staining is visible too. The arrow points to the apical membrane. Scale bar, 50 μm. (b) Immunohistochemical staining of PCFT in human choroid plexus specimen illustrates a uniformly intracellular localization with a punctate pattern. The arrow points to the apical membrane. (c) Immunofluorescence microscopy of human choroid plexus specimen demonstrates distinct distributions of PCFT (red) and FRα (green), as well as minor colocalization. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The arrow points to the apical membrane. Scale bar, 50 μm. (d–k) Rat choroids plexus Z310 cells stably transfected with human FRα and human PCFT were fixed and prepared for immunofluorescence or electron microscopy. Confocal fluorescent microscopy of transfected Z310 cells reveals punctate PCFT (red) and FRα (green) staining (d) that colocalize only marginally. However, FRα (red) significantly colocalizes with CD63 (green), an endosomal marker that is characteristic of MVBs (e), frames indicate the magnified area. Scale bars, 5 μm. Electron microscopy after immunogold labelling of FRα demonstrates enrichment of gold particles within MVBs and close to the fusion point between MVB (black arrows) and plasma membrane (white arrows) (f). Scale bar, 500 nm. Higher magnification reveals that FRα is associated with small ILVs inside the MVB or with vesicles outside the cell (black arrowheads), (g) scale bar, 250 nm and (h) scale bar, 50 nm. Vector-tansfected Z310 cells contain similar ILV (black arrowhead) within MVBs (black arrows) but lack FRα gold staining (i), scale bar, 500 nm. PCFT staining discloses gold particles associated with medium-size electron-dense vesicles (black arrowhead) that are neither located within MVB nor fuse with the plasma membrane (white arrow), (j) scale bar, 250 nm and (k) scale bar, 100 nm. The specificity of the applied antibodies is illustrated in Supplementary Fig. S1.