Cw0130

Immunohistochemistry for ID1, ID2, ID3, and E-cadherin was performed on the TMAs, using a two-step protocol (EnVision™ Detection Systems, Dako, Glostrup, Denmark). The TMAs were dewaxed with xylene, gradually hydrated with gradient ethanol, and washed with phosphate-buffered saline (PBS). Antigens were retrieved by boiling the TMAs in citrate buffer (pH 6.0) at 121°C for 15 minutes. Endogenous peroxidase was blocked by incubating the TMAs in 3% H₂O₂ solution. Then, the slides were washed in PBS for 10 minutes, blocked with 10% normal goat serum (CW0130; CWBIO, Beijing, People’s Republic of China) for 30 minutes at 37°C, and incubated with Anti-ID1 (ID1 RabMab^®; Epitomics, Inc., Burlingame, CA, USA), anti-ID2 (ID2 [D39E8] Rabbit mAb; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-ID3 (Abgent, San Diego, CA, USA), anti-E-cadherin (BD Biosciences Pharmingen, San Diego, CA, USA) at 4°C overnight. The antibody dilution was 1:200 for the ID antibodies and 1:400 for E-cadherin antibody. The immunoreactive products were visualized by the catalysis of 3, 3-diaminobenzidine by horseradish peroxidase (EnVision™ Detection Systems), following extensive washings. The TMAs were then counterstained with Gill hematoxylin (BA-5021, Zhuhai, People’s Republic of China) and dehydrated in ascending grades of ethanol, and finally, cleared in xylene and mounted under a coverslip.

The kidney tissue was cut into small fragments with scissors, and RIPA lysis (P0013, Beyotime Biotechnology) was used to lyse the tissue on ice, after which the tissue was ground with a grinder to make it fully lysed. The supernatant was obtained after high-speed refrigeration centrifugation, and the protein concentration was determined by a BCA kit (CW0130S, CWBIO, Beijing, China). Electrophoresis was carried out through 8–15% SDS-PAGE, and the sample was transferred to PVDF membranes, then blocked at room temperature for 1 h, and subsequently incubated with the primary antibody at 4 ℃ in incubators overnight. The membranes were washed with PBS and then incubated in the secondary antibody at room temperature for 1 h. The membranes were examined using a chemiluminescence system (Beyotime Biotechnology). The following primary antibodies were used: Nrf2 (PA5-27882), HO-1 (PA5-77833), Bcl-2 (PA5-27094), Bax (MA5-14003), and GAPDH (MA5-15738-D680, Thermo Fisher Scientific).