S169984 2

Tissue from IR and BSE negative samples were fixed in 10% neutral buffered formalin, processed, embedded in paraffin blocks and sections were cut at 3-5 µm. Slide mounted tissues were autoclaved in a target retrieval solution (Dako, S169984-2), blocked in 2% normal goat serum and incubated overnight at 4°C with primary antibody 6H4 (Prionics, 01-010). The Envision + HRP conjugated polymer kit (Dako, K400611-2) was used for colorimetric detection of bound primary antibody. Slides were evaluated for the presence of immuno-labeling characteristic of prion infection using light microscopy.

All the mice were sacrificed immediately following the last imaging session. The tumors were then harvested, cut into small pieces of approximately 2 mm in thickness and immersed in 10% neutral buffered formalin for 24 hours. Tumor tissues were embedded in paraffin and sectioned (8 µm thickness) and stained with either cleaved-caspase-3 antibody to measure apoptotic cells or Na⁺/K⁺-ATPase (ab76020, Abcam) to delineate cell membranes. Briefly, tissue samples were de-paraffinized, rehydrated, and antigen retrieval was performed using pH 6.1 citrate buffer (S169984-2, Dako) for 20 minutes at 105 °C in a pressure cooker followed by a 10 minute bench cool down. Samples were treated with 3% hydrogen peroxide, and blocked for 30 minutes in PBS/3% bovine serum albumin/10% donkey serum. Primary Na⁺/K⁺-ATPase antibody was incubated overnight at 4 °C followed by Cy7 conjugated anti-rabbit secondary antibody and DAPI. Caspase-3 and Na⁺/K⁺-ATPase stained slides were scanned by a Leica SCN400 Slide Scanner and an Aperio Versa 200 Slide Scanner, respectively, at 20x to generate high-resolution digital images.