Fig. 4

A Biochemical analysis of PrP^Sc in the brains of diseased TgVole 1 × animals inoculated with spontaneously misfolded recombinant prions Ust01, Ust02, Ust06, and Ust09. 10% homogenates of brains from 3 representative TgVole animals at terminal stage of disease after the intracerebral inoculation with recombinant preparations are shown after proteinase K digestion (85 µg/ml at 42 °C and 450 rpm for 1h) and Western blotting (mAb 9A2, 1:4000). Following inoculation with recombinant preparations, all TgVole 1 × animals exhibited a classical three-banded pattern of PK-resistant PrP^Sc. Some differences in the molecular weight of the unglycosylated band, apparently lower in Ust06 and Ust09 inoculated animals in both the first and second passage. To facilitate distinction of the size differences on the band corresponding to unglycosilated PrP^res, some of the samples were selected for a longer electrophoresis run (small membranes in the right) that allowed further separation and confirmed the strain differences (indicated by black arrows). SSBP/1 sheep scrapie isolate was included as a reference for a classical brain-derived prion strain, and undigested normal brain homogenate (NBH) from TgVole 1 × served as a size reference for full length PrP^C. MW: Molecular weight marker. B Histopathological analysis of spongiosis and PrP^res accumulation in the brains of diseased TgVole 1 × animals from the first passage inoculated with spontaneously misfolded recombinant prions Ust01, Ust02, Ust06, and Ust09. Formalin-fixed paraffin embedded half-brain sections from TgVole animals at the terminal stage of disease were examined. Sections from different brain regions, including parietal cortex (Pc), striatum (S), hippocampus (H), medulla oblongata (Mobl), and cerebellar cortex (Cc) were stained with Hematoxylin–eosin staining (H&E) to assess vacuolation, and with mAb 2G11 (1:1000) to detect misfolded PrP (PrP^res). Vacuolation and PrP^res accumulation, both indicative of prion disease, were observed in various brain areas with distinct patterns for each inoculum, confirming the authentic prion nature of the recombinant preparations. Supplementary figures provide detailed lesion profiles for each potentially distinct conformers, classified according to their electrophoretic mobility patterns after PK digestion. C Histopahtological analysis of spongiosis and PrP^res accumulation in the brains of diseased TgVole 1 × animals from the second passage. TgVole 1 × animals were inoculated with 1% brain homogenates from diseased TgVole animals selected based on their incubation time and PrP^Sc signal intensity. Similar to the first passage, histopathological analysis revealed vacuolation and PrP^res accumulation in all animals, demonstrating the transmissibility of the prion disease induced by the recombinant prions. Detailed lesion profiles for each of the recombinant preparations can be found in Additional file 1: Fig. S6