Tesee precess 48tm homogenizer

A total of 175 mg of whole brain tissue was homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (w/v) by using a TeSeE^TM Precess 48^TM homogenizer (Bio-Rad) following the manufacturer’s instructions. Homogenates were pressed through 0.4 mm needles of a calibration syringe and immediately frozen at −20 °C. To determine the presence of PrP^res, 20 μl of 10% brain homogenates were analyzed by WB as described above. For immunoblotting, membranes were incubated with Sha31 mAb^{19 (link)}.

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).

Brain tissue was homogenized in 5% glucose in distilled water in grinding tubes (Bio-Rad) and adjusted to 10% (w/v) by using a TeSeE™ 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 WB as previously described^{14 (link)}. For immunoblotting, membranes were incubated with Sha31 PrP monoclonal antibody (epitope 148-YEDRYYRE-155 of the goat PrP sequence)^{26 (link)} at a final concentration of 1 μg/mL. Immunocomplexes were detected with horseradish peroxidase- conjugated anti-mouse IgG (Amersham Pharmacia Biotech) 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, 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).