Th f 11 antibody

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).

Immunohistochemistry assay was performed, as in our previous study (Zhang et al., 2017 (link)). Briefly, brain tissue samples were embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, CA, USA) and stored at −80°C. Samples 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 then incubated in 5% BSA for 10 min. Sections were incubated overnight at 4°C with primary antibodies as follows: TH (F-11) antibody (1:50; Santa Cruz Biotechnology Inc.), nitrotyrosine (11C2) antibody (1:50; Santa Cruz Biotechnology Inc.), glial fibrillary acidic protein (GFAP) antibody (1:50; Santa Cruz Biotechnology Inc.) and ionized calcium-binding adapter molecule 1 (IBA-1) antibody (1:500; WAKO, Osaka, Japan). After washing 3 times with PBS for 5 min each, sections were incubated sequentially in HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) for 2 h at 37°C. Sections were visualized with a 3,3-diaminobenzidine (DAB) peroxidase substrate kit (Boster, Wuhan, China). Integrated option density (IOD) was determined using an Image-Pro Plus 6.0 photogram analysis system (IPP 6.0; Media Cybernetics, Bethesda, MD, USA).