Gel pro analyzer v4

Immunohistochemical staining for CENPU and MRPS28 was performed on paraffin-embedded tumors that were fixed in a 10% formaldehyde solution. After dewaxing, hydration, and antigen retrieval, the endogenous peroxidase activity was quenched using H₂O₂ (3%) and goat serum was used to block tumor tissue sections for 30 min at 4°C. After three PBS washes, Triton X-100 (0.5%) was added onto the tissue sections, followed by a 2 min incubation on ice. Sections were washed three times in PBS. A rabbit anti-mouse CENPU antibody or MRPS28 antibody (1:100 dilution, Wuhan Boster Bio-Engineering Co., Ltd., Wuhan, China) was added and the sections were incubated for 12 h incubation at 4°C. After incubation with the primary antibodies, sections were probed using biotinylated secondary antibodies for 2 h followed by three times washing with PBS. Sections were stained using 3,3’-diaminobenzidine color reaction and counterstained with hematoxylin. Following ethanol dehydration, sections were sealed with neutral resin and the integrated optical density (IOD) was analyzed using the Gel-Pro Analyzer V4.0 image analysis software system (Media Cybernetics, Rockville, MD, USA). Large IOD/Area value indicated more protein expression level.

The expression of Nrf2, related factors, and MAPKs in HSC-T6 cells was investigated using Western blot, as described previously[16 (link)]. Briefly, the cells were harvested and re-suspended in a Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, CA, United States) or RIPA lysis buffer [20 mmol/L Tris, 150 mmol/L NaCl, 1% (v/v) Triton X-100, 1% (w/v) digestive phosphatase inhibitors, 1% (w/v) protease inhibitors, 1% (w/v) phenylmethyl sulfonylfluoride (PMSF), pH 7.5] (Sigma-Aldrich). The protein content was determined using a commercial protein reagent kit (Bio-Rad, Hercules, CA, United States). Equal amounts of proteins in each sample were resolved by 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, Sigma-Aldrich) electrophoresis and the proteins were transferred onto PVDF membranes (Sigma-Aldrich). After blocking with skim milk, the membranes were incubated with the specific antibodies (Table 2) for 24 h at 4 °C. After washing, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Pierce Biotechnology, Rockford, United States) for 2 h at 37 °C. Proteins were detected with an all-enhanced chemiluminescence detection system (Syngene, United Kingdom) and quantified using a Gel-Pro Analyzer v4.0 (Media Cybernetics, L.P., United States). β-actin was used as the loading control.