Anti versican antibody

GSE30122 was utilized as a validation array to further verify the hub gene. Paraffin-embedded kidney sections were collected from 6 healthy living transplant doners and 6 DKD patients who were diagnosed with pathology in 2021. The experiment protocols were approved by the Research Ethics Committee of The First Affiliated Hospital, College of Medicine, Zhejiang University. Kidney paraffin sections from patients with DKD and LDs were stained with versican. Briefly, paraffin-embedded sections were dewaxed, incubated in citrate buffer at 95–98°C for 10 min for antigen retrieval, followed by in 0.3% H₂O₂ for 30 min at room temperature to block endogenous peroxidase in blocking buffer (5% bovine serum albumin) for 30 min to block non-specific binding, and then incubated with anti-versican antibody (Abcam, Cambridge, United Kingdom, ab19345, 1:150) overnight at 4°C, followed by incubation with secondary antibody for 30 min at room temperature. The DAB substrate solution was applied to visualize the color of primary antibody staining. The sections were counterstained with hematoxylin, dehydrated, vitrified, and sealed with neutral balsam.

DPCs were seeded in a 24-well plate at a density of 3 × 10⁴ and cultured for 24 h. Then, Hordenine at the concentration of 0, 25 and 50 µmol/L was added to treat DPCs for 24 h. Then, cells were washed with PBS for three times, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and blocked with 1% FBS for 0.5 h. The cells were then incubated overnight at 4 °C with the specific primary antibodies, followed by incubation for 2 h with fluorescent secondary antibody and staining with DAPI for 5 min. Photographing were conducted under an Olympus microscope. All the primary antibodies used in immunofluorescence staining were as follows: 1:200 anti-Ki67 antibody (Abcam, 16667, Cambridge, UK), 1:200 anti-β-catenin antibody (Proteintech, 51067-2-AP, Chicago, IL, USA), 1:100 anti-ALP antibody (Abcam, 65834, Cambridge, UK), 1:100 anti-Versican antibody (Abcam, 19345, Cambridge, UK).