Figure 3.

Detection of PrP^Sc in the brain, spleen and granuloma of 127S-infected mice. At various times post-infection, groups of animals were sacrificed and brain, spleen and granuloma were removed for tissue processing and analysis. (A) proteinase-K digestion followed by western blot analysis using biotinylated Sha31 anti-PrP antibody. Note that the exposure times for brain and spleen WB membranes ranged from 3 to 5 minutes, while the granuloma samples required longer exposure times (over 3 hours). (B) Immunofluorescence (IF) detection of PrP^Sc in the spleen or granuloma of tg338 mice inoculated with 127S prions. Labeling of PrP^Sc in spleens (B-1, B-3) and granulomas lymphofollicular structures (B-2, B-4) from control (B-1, B-2) or infected animals (B-3, B-4). Ten-micrometer-thick slices were cut on a cryostat, fixed in 5% paraformaldehyde, permeabilized in 0.5% X-100 Triton, and treated with 3.5 N guanidinium thiocyanate before the avidin-biotin blocking reaction followed by biotinylated Sha31 incubation and streptavidin-Alexa-488 labeling, slide mounting using fluoromount and acquisition under a mono CCD camera. Images in false colors: blue, DAPI counterstaining; green; biotinylated Sha31, labeling guanidinium-resistant PrP^Sc. Bars: 20 µm. (C) PMCA templating activity of PrP^Sc present in individual brains, black ■; spleens, red ●; and granulomas, blue ▲ from 127S-inoculated mice. The log titer (regarded as the last Log₁₀-positive dilution) is represented versus time (days post-inoculation). Dotted lines represent the detection threshold. Gray squares represent the inoculum titer. See Supplementary Table 1 for a percentage of positive samples.