Fig. 3. Expression of PrP^C in brain endothelial cell lines. (A) Immunofluorescence labeling of PrP^C in rat (RBE4) and human (hCMEC/D3) brain endothelial cell lines. Cells were incubated with anti-PrP^C (SAF32, 2 µg/ml) monoclonal antibodies for 16 hours at 4°C, then with Cy2-conjugated anti-mouse antibodies for 45 minutes at 25°C. Images were collected with a confocal fluorescence microscope. Bars, 20 µm. (B) RT-PCR detection of PrP^C transcripts in rat (RBE4) and human (hCMEC/D3) brain endothelial cells; primary rat astrocytes (Astro) and the human U373 astrocytoma line (U373) were used as positive controls, respectively. Rat HPRT or human GAPDH were used for standardization. The size of the transcripts (rat: 244 bp; human: 243 bp) was as expected. Control lanes show no amplified fragments in the absence of cDNA.