Ab169782

Liver tissues were fixed with 10% buffered formalin for 48 h, embedded in paraffin, and cut into 4-6 μm slices. After dehydration with ethanol, incubation with citrate buffer (pH 6.0), and pretreatment with 3% hydrogen peroxide (H 2 O 2 ), slices were incubated with primary antibody against alpha-smooth muscle actin (α-SMA) (1:400, MA5-11547, Ther-moFisher), collagen type I (Col1α1; 1:500, AF7001, Biosciences), glutathione peroxidase 4 (Gpx4; 1:250, ab125066, Abcam), and prostaglandin endoperoxide synthase 2 (Ptgs2; 1:100, ab169782, Abcam) overnight at 4 • C. Slices were then incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, #31460, ThermoFisher) and were visualized with diaminobedzidine.

The ARPE-19 cells in each group were collected and digested using RIPA Lysis Buffer containing phenylmethylsulfonyl fluoride (PMSF; 80 μL) for 30 min. The supernatant was collected in a high-speed bench refrigerated centrifuge. The concentration of protein was detected using the BCA method. After SDS-PAGE electrophoresis, the protein was transferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked using bovine serum albumin (BSA) for 1 h and then washed with Tris-buffered saline with Tween (TBST) 3 times. The primary antibody (NF-κB p65, ab32536, 1 : 30; COX-2, ab169782, 1 : 2000; Bcl-2, ab32124, 1 : 1000; Bax, ab182733, 1 : 2000; cleaved caspase-3, ab32042, 1 : 500; GADPH, ab8245, 1 : 2000; all from Abcam, Cambridge, UK; and cleaved PARP, #9541, 1 : 1000; Cell Signaling Technology, Inc., Danvers, USA) was produced using the corresponding dilutions detailed above, added to the membrane and incubated at 4°C overnight. The membrane was then washed with TBST 3 times on the 2 nd day. The secondary antibody conjugated with horseradish peroxidase (HRP; ab7090, ab97040, 1 : 10000; Abcam) was then added and incubated with the membrane in a shaker at room temperature for 1 h. GAPDH was used as an internal reference.