Immunohistochemistry Protocol for TH and 4-HNE

Immunohistochemistry was performed as in our previous study^{64 (link)}. Briefly, sections were cut into 10-μm slices and antigen retrieval was performed using citrate buffer. Sections were treated with 3% hydrogen peroxide (Sangon Biotech Co., Ltd., Shanghai, China) in PBS for 10 min and were then incubated in 5% BSA for 60 min. Sections were incubated overnight at 4 °C with primary antibodies as follows: TH (F-11) antibody (1:50; Santa Cruz Biotechnology Inc.), and 4-HNE antibody (1:50; R&D Systems.). After washing three times with PBS for 5 min each wash, sections were incubated sequentially in HRP-conjugated goat anti-mouse secondary antibody (ZSGB-BIO, PV6000, Beijing, China) for 1 h at room temperature. Sections were visualized with a 3,3-diaminobenzidine peroxidase substrate kit (ZSGB-BIO, Beijing, China). Integrated optical density was determined using an Image-Pro Plus 6.0 photogram analysis system (IPP 6.0; Media Cybernetics, Bethesda, MD, USA).