3 m guanidine thiocyanate

The intensity and distribution of PrP Sc deposition was evaluated using the paraffin-embedded tissue (PET) blot method, as previously described [25] (link). Sections from paraffin-embedded brains (4-μm thick) were collected on a nitrocellulose membrane (0.45-μm pore size; Bio-Rad, Richmond, CA) and dried at 55°C for 24 h. After deparaffination and rehydration, sections were digested for 2 h at 56°C with 250 μg/ml proteinase-K (PK) (Applied Biosystems) in PK digestion buffer containing TBS (Tris-buffered saline) and 0.1% Brij 35P (Sigma-Aldrich). After washing with TBST (Tris buffered saline; 0.05% Tween 20), membrane-attached proteins were denatured in 3 M guanidine thiocyanate (Sigma-Aldrich). Sections were then blocked with 1% casein in TBST and incubated with Sha31 primary monoclonal antibody (1:8000; SPI-Bio). After incubation with an alkaline phosphatase-coupled goat anti-mouse antibody (DAKO) immunostaining was visualized using NBT/BCIP (Nitro blue tetrazolium /5-bromo-4-chloro-3indolyl-phosphate; Sigma-Aldich). PrP Sc deposits were evaluated semi-quantitatively, as described for spongiform lesions, using a Zeiss Stemi DV4 stereo microscope.

Tg338 brains fixed in formalin were embedded in paraffin wax, cut into 4-µm-thick sections, and mounted on glass slides for morphological evaluation using hematoxylin and eosin (HE) staining. Analysis of PrP Sc deposition was performed using the paraffin-embedded tissue (PET) blot method as previously described [34] . Briefly, paraffinembedded sections (4 µm) were collected onto nitrocellulose membranes (0.45 μm pore size; Bio-Rad) and digested with 250 μg/ml of proteinase K for 2 h at 56 °C. Membraneattached proteins were denatured with 3 M guanidine thiocyanate (Sigma-Aldrich) and PrP Sc was detected using Sha31 mouse monoclonal antibody (1:8000 dilution for 1 h; SPI-Bio). Sections were then incubated with an alkaline phosphatase-coupled goat anti-mouse antibody (1:500 dilution for 1 h; Dako) and enzymatic activity was visualized using NBT/BCIP chromogen (Sigma-Aldrich).