Cy1851

After the cell fixation and permeabilization procedure, the cells were blocked in 5% BSA and subsequently incubated in the anti‐CAV1 (sc‐53564, Santa Cruz) and anti‐NRF2 primary antibodies (CY1851, Abways) overnight at 4°C. On the next day, the cells were washed three times with PBS and incubated in the dark with fluorescence‐labelled secondary antibodies for 1 h. After 3× PBS wash, nuclei staining with DAPI (Sigma‐Aldrich) was conducted for 5 min. For confocal imaging, the fluorescence images were obtained with a confocal laser scanning microscope (Carl Zeiss) and the fluorescence intensity was processed and analysed using ZEISS ZEN Imaging Software (Carl Zeiss).

The molecular changes in CAV1 and NRF2 levels and oxidative stress index (8‐OHdG) levels were measured by immunohistochemistry. After deparaffinization, the tissue sections were soaked in a 3% H₂O₂ solution for 15 min. Subsequently, the sections were washed in PBS and antigen retrieved using 0.1 M sodium citrate. After incubation with 3% bovine serum albumin (BSA) (Sigma‐Aldrich) at 37°C for 30 min, the tissues were added and incubated with antibody to CAV1 (sc‐53564, Santa Cruz), NRF2 (CY1851, Abways), and 8‐OHdG (sc‐66036, Santa Cruz) overnight at 4°C, respectively. Then, the sections were subsequently treated with secondary antibodies at room temperature for 30 min on the next day. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich) and incubated for 10 min. The sections were evaluated with a bright‐field microscope (BioTek). Ten microscopic fields were randomly chosen and counted according to the positive number and staining intensity of each specimen.