Rabbit hrp

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.

Liver histology and liver fibrosis were assessed on paraffin-embedded liver sections by Hematoxylin/Eosin and Sirius Red staining respectively. Ki67 was assessed by immunohistochemistry (IHC) on paraffin-embedded liver sections. Briefly, sections were deparaffinated and antigens were exposed by 30min steamer incubation in Tris-EDTA (pH = 9) buffer. After antigen retrieval procedure, endogenous peroxidase activity was inhibited for 10 min with 3% hydrogen peroxide. Sections were blocked for 1 h with normal goat serum and incubated with anti-Ki67 (1:300; 12202S, Cell Signaling Technology) primary antibody O/N at 4°C. HRP-Rabbit (7074S, Cell Signaling Technology) secondary antibody was added. Color development was induced by incubation with a DAB kit (8059S, Cell Signaling Technology) and the sections were counterstained with hematoxylin. Sections were dehydrated and mounted. Positive cell count was performed using QuPath software.

To examine protein localization and expression, the formalin-fixed tissues were de-paraffinized. After deparaffinization and dehydration of paraffin sections, heat-induced epitope retrieval was performed using a pressure cooker and sodium citrate buffer (10 mM sodium citrate, 0.05% tween-20, pH 6.0). Once boiled, slides were transferred from PBS to the sodium citrate buffer in pressure cooker for 10 min. Slides were then cooled to room temperature for 30 min, and permeabilized with permeabilization buffer containing 0.3% triton-100 in PBS for 10 min. To block endogenous peroxidase activity, slides were incubated in 3% hydrogen peroxide for 10 min and blocked with animal-free blocking solution (Cat# 15019; Cell Signaling, Inc.). After blocking, slides were incubated overnight at 4°C with mouse monoclonal anti-TGF-β₁ (1:50; Cat# sc-130348; Santa Cruz, Inc.) or rabbit polyclonal anti-TGF-β₁ receptor (1:100; Cat# ab235178; Abcam, Inc.) and subsequently incubated with SignalStain Boost Detection Reagent (HRP mouse; Cat# 8125; or HRP Rabbit; Cat# 8114; Cell Signaling, Inc.) for 30 min at room temperature. Slides were then incubated for 2–10 min with SignalStain DAB (Cat# 8059; Cell Signaling, Inc.), immersed in distilled H₂O, stained with hematoxylin (Cat# 14166; Cell Signaling, Inc.) and mounted with coverslips. All washing steps were done three times with PBS-T (tween-20, 0.05%).

Mineralocorticoid Receptor (MCR): Cochlear section slides were washed in TBS twice for 5 min. They were then incubated for 10 min at room temperature in 3% H₂O₂, diluted in methanol, and washed with TBS for 2 × 5 min. Each section was treated with 100-400 μl blocking solution (0.3% triton- X 100 and 5% normal goat serum in TBS) for 1 hr at room temperature. Then removed from the blocking solution, with the addition of 100-400 μl anti-MCR (SC-25709, Santa Cruz Biotechnology, Dallas, TX) 1:25 diluted in blocking solution. Next, incubated overnight at 4°C. In the morning, the slides were washed in TBS 3 × 5 min, then Covered with 1-3 drops of signal stain boost IHC detection reagent (HRP, Rabbit, Cell Signaling, Danvers, MA) and incubated in a humidified chamber for 30 min at room temperature. Then, 3 × 5 min washes with TBS. Then 100-400 μl DAB was placed on each slide for intensification, then the slides were immersed in dH₂O, and washed 2 × 5 min, followed by dehydration and coverslipping.