Avidin biotin hrp complex reagent solution

The mice were subjected to transcardial perfusion, their brains were removed and cryotome sections were collected. Every fifth section from the region between bregma -1.82 mm and -2.18 mm (according to Paxinos and WatsonMouse Brain Atlas) was analyzed. For DNA hydrolysis, tissue sections were washed twice in phosphate-buffered saline, incubated in 2 N HCl for 30 min at 37°C, following neutralization in borate buffer (0.1 M, pH 8.5) for 15 min. Immunohistochemistry (IHC) was performed with specific anti-BrdU primary antibodies (Oxford Biotechnology, Oxford, UK). After incubation with anti-BrdU, sections were incubated with the biotinylated secondary antibody for 1 h at 25 °C and then with avidin-biotin-HRP complex reagent solution (Vector Laboratories, Burlingame, CA, USA). The peroxidase reaction was finally performed using diaminobenzidine tetrahydrochloride (Vector Laboratories). Digital images of IHC staining were captured using a Leica DM750 microscope. Every fifth section was taken from the region between 1.46 to 2.80 mm posterior to bregma. BrdU-positive cells were quantified.

For immunohistochemical (IHC) staining, sections were treated with 3% H₂O₂ and 4% BSA to inactivate endogenous peroxidation and block non-specific binding, respectively. The sections were incubated with a primary antibody against tyrosine hydroxylase (TH) or Iba-1 (1:1000) overnight, then with biotinylated secondary antibodies for 1 h at 25 °C room temperature, followed by an avidin-biotin-HRP complex reagent solution (Vector Laboratories, Burlingame, CA, USA). Subsequently, the peroxidase reaction was performed using diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescence (IF) staining, sections were treated to block non-specific binding and were incubated with primary antibodies, followed by secondary antibodies conjugated to a fluorophore. Specific details of antibodies used in IHC and IF staining are summarized in Table S2. Digital images of the IF staining were captured using a Leica DM750 microscopy and quantified using Image J. Four to six sections per brain were stained and quantified. The number of target protein-positive cells per area (mm²) was counted, and the co-localization rate (%) was calculated using the following formula: {(the number of target protein-positive cells co-stained with cell type marker)/the number of cell type marker-positive cells} × 100.

40-μm-thick coronal sections were cut with a cryotome (CM1860; Leica, Mannheim, Germany) and stored at −20 °C in an anti-freezing solution (30% ethylene glycol and 30% glycerol in phosphate-buffered saline). Sections were treated with 0.3% H₂O₂ for 30 min and 4% BSA for 1 h before IHC staining to inhibit endogenous peroxidation and prevent non-specific binding, respectively. Sections were incubated with primary antibodies overnight, then with biotinylated secondary antibodies for 1 h at 25 °C before being incubated with avidin–biotin–HRP complex reagent solution (Vector Laboratories, Burlingame, CA, USA). Following that, a peroxidase reaction was carried out using diaminobenzidine tetrahydrochloride (Vector Laboratories). BV2 cells were fixed with 4% paraformaldehyde for 15 min before being incubated with 2% BSA/3% horse serum for 30 min to block nonspecific antibody binding. The cells were then incubated with anti-HNE primary antibodies overnight, followed by fluorochrome-conjugated secondary antibodies (Alexa Fluor 488) for 1 h at 25 °C. A Leica DM750 microscope was used to capture digital images of the IHC and IF staining, and ImageJ software, version 1.37, was used for quantification (National Institutes of Health, Bethesda, MD, USA).