PrP^Sc is present both in and around MadCam1-positive HEVs in <i>TNFR1^−/−</i>-Ig mesenteric lymph nodes.

<p>TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig were sacrificed at 60 d.p.i. Immunofluorescence (A–I) and histoblots (J & K) were then performed on frozen sections from prion-infected TNFR1^−/−-Ig mesenteric lymph nodes. Co-IF with anti-serum (XN) against PrP (green; A) and MadCam1 (red; B) showed points of intense PrP immunoreactivity localized to HEVs (C). Confocal co-IF with the amyloid-binding dye, p-FTAA (green; D) and MadCam1 (red; E,F) revealed some points of PrP^Sc association with HEVs (F); however much of the PrP^Sc was present outside of HEVs (I). Histoblots pre-stained with PNAd antibody and developed with AP (pink; J) also revealed some prion-infected HEVs (black arrows), some non-infected HEVs (white arrow), and some PrP^Sc deposits that were not HEV-associated (yellow arrow). (K) Total numbers of PNAd-positive HEVs in histoblot co-stains were counted and scored as PrP^Sc-positive (PrP^Sc+; black) or PrP^Sc-negative (PrP^Sc+; white), and total PrP^Sc deposits were counted and scored as PNAd-positive (PNAd+; black) or PNAd-negative (PNAd; white). 35% of HEVs were PrP^Sc-positive, and 58% of PrP^Sc deposits were PNAd-positive. Size bars in A–F = 50 µm. Size bars in G–I = 100 µm.