Anti mouse 4 hne

We applied IHC analysis against 4-hydroyneanel (4HNE) to detect lipid peroxidation in the liver tissue. Liver sections (4 µm thick) were prepared using deparaffinizing and rehydrating progressions and microwaving in an antigen retrieval buffer (sodium citrate 10 mM, pH 6.0) for 5 min. After cooling and washing, we added 3% H₂O₂ in methanol to remove erythrocytes. Then, we incubated the blocking buffer for 1 h at RT and applied anti-mouse-4-HNE (R&D system, MA, Cat. #MAB3249, 1:200) overnight at 4 °C. The next day, after washing, we applied a secondary antibody and developed the positive signals using 3,3′-Diaminobenzidine (DAB) after amplifying the HRP (ImmPACT^® DAB Substrate, Cat. #SK-4105). We used ICH, a commercially available product, as the reagent (VECTASTAIN^® ABC Kits, Burlingame, CA, USA). A TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was performed using a commercial kit (Sigma-Aldrich Chemical Co., St. Louis, MO, USA).

We purchased manganese chloride (MnCl₂, Cat. No. 203734), vanadium pentoxide (V₂O₅, Cat. No. 204854), protease cocktail, and phosphatase inhibitors (Cat. No. 1861281) from Sigma (St. Louis, MO, USA). The Bradford protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Perchloric acid (Cat. No. 244252), EDTA (Cat. No. 79884), DA (Cat. No. PHR1090), and DOPAC (Cat. No. 11569) were purchased from Millipore Sigma (Burlington, MA, USA). NaS₂O₅ (Cat. No. S244) was purchased from Fisher (Hampton, NH, USA). The primary antibodies used in this study were as follows: anti-mouse tyrosine hydroxylase (TH) obtained from Millipore (Billerica, MA, USA, Cat. No. MAB318); the anti-mouse glial fibrillary acidic protein (GFAP) purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-mouse 4HNE acquired from R&D Systems, Inc. (Minneapolis, MN, USA, Cat. No. MAB3249); anti-mouse α-synuclein (Cat. No. 610787) and anti-mouse native PKCδ (Cat. No. 610397) obtained from BD Biosciences (Palo Alto, CA, USA); anti-rabbit phospho-PKCδ purchased from Invitrogen (Waltham, MA, USA, Cat. No. PA5–17903); and anti-β-actin purchased from Sigma (Cat. No. A5441). The Alexa 680-conjugated anti-mouse and IRDye800-conjugated anti-rabbit secondary antibodies were purchased from Invitrogen and Rockland Inc., respectively.