Chemidoc wrs system

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).

We homogenized frozen brain tissues (175 + 20 mg) in 5% glucose in distilled water in grinding tubes (Bio-Rad, https://www.bio-rad.com) adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad). We determined the presence of PrP^res in transgenic mouse brains by using WB, using the reagents of the ELISA commercial test TeSeE (Bio-Rad). We prepared brain homogenates (10–100 µL of a 10% [wt/vol]) as previously described (18 (link)) and loaded samples into 12% Bis-Tris Gel (Criterion XT; Bio-Rad). We transferred proteins electrophoretically onto polyvinylidene fluoride membranes (Millipore, https://www.sigmaaldrich.com) and blocked overnight with 2% bovine serum albumin blocking buffer. We incubated membranes with Sha31 (25 (link)) mAb at a concentration of 1 µg/mL. Sha31 recognizes the 145-WEDRYYRE-152 epitope of the human-PrP^C sequence, which is conserved in sheep and bovine sequences. We detected immunocomplexes by incubating the membranes for 1 h with horseradish peroxidase conjugated antimouse IgG (GE Healthcare Amersham Biosciences, https://www.gelifesciences.com). We developed immunoblots with enhanced chemiluminescence in ECL Select (GE Healthcare Amersham Biosciences) and captured images by using the ChemiDoc WRS+ System and processed them by using Image Lab 5.2.1 software (both Bio-Rad).

Western blotting analysis was done as previously described [23 (link)]. Briefly, 175 + 20 mg of frozen mouse brain tissue were homogenized in 5% glucose distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (wt/vol) by using a TeSeE Precess 48TM homogenizer (Bio-Rad). To detect brain PrP^res 100 μL of 10% (wt/vol) brain homogenate were subjected to digestion with 40 μg/mL of PK in buffer 5% sarkosyl, 5% Triton X100, 1 M Urea, and 16 mM Tris–HCl (pH 9.6) at 60 °C for 15 min. Digested samples were loaded samples into 12% Bis-Tris Gel (Criterion XT; Bio-Rad). After electrophoretic transference of proteins onto polyvinylidene fluoride membranes (Millipore) and blocking overnight with 2% bovine serum albumin blocking buffer, membranes were incubated with 12B2 [24 (link)] and Sha31 [25 (link)] mAb at a concentration of 1 μg/mL. 12B2 recognizes the 89-WGQGG-93 epitope of the human-PrP^C sequence while Sha31 recognizes the 145-WEDRYYRE-152 epitope of the human-PrP^C sequence. Immunocomplexes were detected by 1 h membrane incubation with horseradish peroxidase conjugated antimouse IgG (GE Healthcare Amersham Biosciences) and development with enhanced chemiluminescence in ECL Select (GE Healthcare Amersham Biosciences). Images were captured using the ChemiDoc WRS+ System and processed them by using Image Lab 5.2.1 software (both Bio-Rad).