0.45 μm pore nitrocellulose membranes

Pet-blot was used for the evaluation of PrP^Sc distribution in the brains. We have previously shown the PET-blot images of three of six strains, namely sCJD-MV1, GSS-A117V, and CWD [36 (link),41 (link),44 (link)]. In this study, in order to compare the PrP^Sc distribution among all strains, we performed the PET-blot of previously published strains and those included in this work. For each strain, three vole brains were analyzed.
Brain sections of 6 μm were collected on 0.45-μm-pore nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) and dried at 37 °C for two days. After deparaffination and hydration, sections were treated with proteinase K (50 μg/mL) overnight at 55 °C. Next, proteins on the membranes were denatured using guanidine isothiocyanate 4 M for 15 min. After incubation with blocking solution for 60 min, sections were incubated with mouse antibody SAF84 (Alfatech; Auckland, New Zealand, 1:200) for 90 min. Following washes in TBS with 0.1% Tween-20, a rabbit anti-mouse alkaline phosphatase-conjugated antibody was applied for 90 min. After washing in TBST, membranes were adjusted to alkaline pH by incubating two times in NTM (Tris-HCl 100 mmol/L, NaCl 100 mmol/L; MgCl₂ 50 mmol/L). The antibody reaction was visualized by formazan reaction using NBT/BCIP.

We embedded brain and lymph node samples in paraffin wax, sectioned at 5
μm, and stained with hematoxylin and eosin or subjected to
immunohistochemical or paraffin-embedded tissue blot analysis. We pretreated
sections for immunohistochemistry with 98% formic acid for 5 min, followed by
autoclaving in citrate buffer for 5 min at 121°C. We then treated
sections with 6% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in
phosphate-buffered saline for 60 min. We performed immunohistochemical detection
of PrP^Sc with L42 monoclonal antibody (mAb) (R-Biopharm, Darmstadt,
Germany) at 0.01 μg/mL in phosphate-buffered saline overnight at
4°C. We treated sections with secondary biotinylated mouse antibody
(Vector Laboratories), ABC Complex (Vector Laboratories) for 45 min, and
diaminobenzidine (Sigma-Aldrich, St. Louis, MO, USA) for 3 min. We used
Mayer’s hematoxylin for counterstaining. Each run comprised positive- and
negative-control sections. We analyzed 3 sections from each lymph node
sample.
We collected sections for paraffin-embedded blot on prewetted
0.45-μm–pore nitrocellulose membranes (Schleicher & Schuell,
Dassel, Germany) and dried membranes for 24 h at 55°C. We performed
membrane treatments, proteinase K (PK) (Sigma-Aldrich) digestion (50
μg/mL), and immunodetection as described (15 (link)). We used mAb L42 (0.01 μg/mL) as the
primary antibody.