Anti p53

Paraffin-embedded mouse kidney sections (3 µm thickness) were prepared by routine procedures. The sections were stained with periodic acid-Schiff (PAS) reagents by standard protocol. For immunohistochemical staining, kidney sections were deparaffinized with xylene, and then gradually rehydrated in ethanol. Hydrogen peroxide (3%) was used to eliminate endogenous peroxidase. Antigen retrieval was performed by microwave using the heat mediated antigen retrieval technique. After blocking with 1% donkey serum for 1 h, the sections were incubated with primary antibodies overnight at 4°C and subsequently washed and incubated with secondary antibodies for 1 h at room temperature. The sections were visualized by using AEC substrate Kit under an Olympus light microscope. The primary antibodies were as follows: anti-Coronavirus nucleocapsid (sc-66012; Santa Cruze Biotechnology), anti-KIM-1 (AF1817; R&D Systems), anti-p53 (A0263; Abclonal), anti-caspase-3 (A2156; Abclonal).

Western Blotting was conducted as previously described (Pang et al., 2017 (link)). Briefly, proteins were extracted from cells and cochlea tissues using radioimmunoprecipitation assay lysis buffer (Thermo plus, USA). Protein samples (20 μg) were resolved on a 10% SDS-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). After being blocked with 5% nonfat milk, the membranes were incubated with anti-DRP1 (1:1,000, Proteintech, Rosemont, IL, USA), anti-p-DRP1 (1:1,000, Abclonal, Woburn, MA, USA), anti-LC3I/II (1:1,000, CST, USA), anti-P62 (1:1,000, CST, USA), anti-P53 (1:1,000, Abclonal, Woburn, MA, USA), and anti-P21 (1:1,000, Abclonal, Woburn, MA, USA) at 4°C overnight, followed by incubation with secondary antibodies (1:3,000) at room temperature for 1 h. The immunoreactive bands were then detected by enhanced chemiluminescence (Millipore, Burlington, MA, USA). Band intensities were analyzed using ImageJ (NIH, USA). β-actin was applied as loading and internal control.