Grinding tubes

Brain tissue was homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (w/v) by using a TeSeETM Precess 48TM homogenizer (Bio-Rad) following the manufacturer’s instructions. To determine the presence of PrP^res in transgenic mouse brains, 100 µL of 10% brain homogenate were analyzed by Western blotting as previously described [7 (link)] with some modifications. Briefly, digestion was done with 40 µg/mL of proteinase K in buffer 5% sarkosyl, 5% Triton X100, 1 M Urea and 16 mM Tris–HCl (pH 9.6) at 60 °C for 15 min. Samples were electrophoresed in 12% Criterion XT Bis–Tris Gel (BioRad). For immunoblotting, membranes were incubated with 12B2 PrP monoclonal antibody (epitope ₉₃WGQGG₉₇ of the sheep PrP sequence) at a final concentration of 1 μg/mL. Immunocomplexes were detected with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Pharmacia Biotech) after incubating the membranes for 1 h, and blots were developed with chemiluminescent substrate ECL Select (GE Healthcare Amersham Biosciences). Images were captured using ChemiDoc XRS + System and then processed using Image Lab 5.2.1 Software.

We homogenized frozen brain tissues (175 ± 20 mg) in 5% glucose in distilled water in grinding tubes (Bio-Rad, Hercules, CA, USA) adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad), according to the manufacturer’s instructions. We determined presence of PrP^res in transgenic mouse brains by Western blot, using the reagents of the ELISA commercial test TeSeE (Bio-Rad). Based on a previously described protocol (31 (link)), we treated 10–100 μL of 10% wt/vol brain homogenates with proteinase K; the resulting samples were loaded in 12% Bis-Tris Gel (Criterion XT; Bio-Rad). We transferred proteins electrophoretically onto PVDF membranes (Millipore, Billerica, MA, USA), which were blocked overnight with 2% BSA blocking buffer (Sigma-Aldrich, St. Louis, MO, USA). For immunoblotting, we incubated with Sha 31 (44 (link)) monoclonal antibody (mAb) at a concentration of 1 µg/mL to identify the 145-WEDRYYRE-152 epitope of the human PrP^C sequence. To detect immunocomplexes, we incubated the membranes for 1 h with horseradish peroxidase conjugated anti-mouse IgG (GE Healthcare Amersham Biosciences, Little Chalfont, UK). Immunoblots were developed with enhanced chemiluminiscence ECL Select (GE Healthcare Amersham Biosciences). Images were captured using the ChemiDoc WRS+ System (Bio-Rad) and processed using Image Lab 5.2.1 software (Bio-Rad).