Pierce protein free t20 blocking buffer

Immediately after ex vivo analysis, RV tissue was then washed in ice‐cold saline and snap‐frozen. RV tissue was then homogenized using an Omni international tissue grinder (Thermo Fisher Scientific, Waltham, MA) in ice‐cold RIPA lysis buffer (Thermo Fisher) containing proteinase inhibitor cocktail (EMD Millipore/Sigma‐Aldrich, St. Louis, MO) and PhosStop inhibitor cocktail (Roche, Indianapolis, IN). After homogenization, lysate was sonicated for ten one‐second pulses at 100% power and then centrifuged. The supernatant was saved and used as whole lung lysate. Protein concentration was measured using BCA Protein Assay (Thermo Fisher). Rabbit polyclonal anti‐phospho‐p38MAPK, anti‐total p38MAPK, anti‐bcl2, and anti‐bax (all used at 1:1000, and from Cell Signaling, Danvers, MA) and mouse monoclonal anti‐Vinculin loading control (1:5000; Calbiochem; Billerica, MA) primary antibodies were used, all diluted in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). All antibodies used have been extensively validated (Lin et al. 2005; Bernal‐Mizrachi et al. 2006; Bikkavilli et al. 2008; Tang et al. 2008; Slone et al. 2011; Choi et al. 2016; Jiang et al. 2016; Zhang et al. 2017). Rabbit‐HRP (Cell Signaling) and mouse‐HRP (KPL, Gaithersburg, MD) secondary antibodies were diluted 1:2000 in Pierce Protein‐Free T20 Blocking Buffer (Thermo Fisher). Densitometry was performed using ImageJ.

The ML C antigens (approximately 10 μg per well) were run on a 12% SDS-PAGE gel with 4% stacking gel at 180 V constant voltage for 50 min. After electrophoresis, the proteins were transferred from the gel to a nitrocellulose sheet (Bio-Rad, Hercules, CA, USA) at 95V for 55 min using Mini-Protean Tetra Cell (Bio-Rad, Hercules, CA, USA). Nitrocellulose strips (NCS) were blocked with Pierce Protein-Free T20 Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA) for one hour at RT, and then washed three times in PBS–Tween buffer (pH = 7.2). Following this, the NCSs were incubated with shaking for one hour at RT with meat juice samples diluted 1:20 in Pierce Protein-Free T20 Blocking Buffer (Thermo Fisher Scientific, Waltham, MA, USA). Afterward, the NCSs were washed and then incubated with shaking for one hour at RT with secondary antibody, i.e., horse radish-peroxidase-conjugated Anti-dog IgG (Sigma-Aldrich, Saint Louis, MO, USA), diluted by 1:25,000. Finally, after washing, the NCSs were developed using SIGMAFAST™ 3,3′-diaminobenzidine (Sigma-Aldrich, Saint Louis, MO, USA) and visualized with the Chemi Doc MP Imaging System (Bio-Rad, Hercules, CA, USA).