Anti lipocalin 2 ngal

The anti-Ly6G antibody (1:200, Cell Signaling), anti-Lipocalin-2/NGAL (1:1000, abcam) were used for IHC. Images were taken using Nikon ECLIPSE Ni-U. Image J software (NIH) was used for process and quantify Ly6G-positive areas in the images.

For preparing the protein sample, HK-2 cells and kidney tissues were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer, and the supernatants were collected after centrifugation at 13,000 g at 4°C for 15 min. Protein concentration was quantified using the BCA Protein Assay (K813-5000-1; BioVision). Proteins were separated with 8%–15% SDS-PAGE and blotted onto PVDF microporous membranes (IPVH00010; Millipore Sigma). The membranes were then blocked with 5% milk at room temperature for 1 h and incubated with primary antibodies at 4°C overnight, followed by incubating with HRP-conjugated secondary antibodies for 1 h at room temperature. The primary antibodies are as follows: anti-mKlotho (AF1819; R&D Systems), anti-PARP (#9532; Cell Signaling Technology), anti-p53 (#2524S; Cell Signaling Technology), anti-FADD (sc-271748; Santa Cruze Biotechnology), anti-cacspase-3 (#9622; Cell Signaling Technology), anti-Coronavirus nucleocapsid (sc-66012; Santa Cruze Biotechnology), anti-Flag (F1804; Sigma-Aidrich), anti-KIM-1 (Ab47635; Abcam), anti-Lipocalin-2/NGAL (ab63929; Abcam), anti-GAPDH (Z1208; Ray antibody Biotechnology). The protein bands were quantified by ImageJ software (NIH) and normalized to the loading controls.