Mouse monoclonal anti tyrosine hydroxylase th

Immunohistochemical staining was performed according to a standard immunoperoxidase protocol for free-floating sections using following antibodies: mouse anti-dopamine- and cAMP-regulated phosphoprotein (DARPP-32; 1:5000; BD Biosciences, USA), mouse monoclonal anti-tyrosine hydroxylase (TH; 1:1000; Sigma, USA), rat anti-human α-syn (aa 116-131 hα-syn; dilution and company please; 15G7, Enzo Life Sciences, Germany), rabbit anti-phosphorylated α-syn (pα-syn; 1:1000; Abcam, UK), and mouse anti-nitrated α-syn (nα-syn; 1:1000; Invitrogen, Zymed Laboratories, USA). In case of the visualization of partially proteinase-K (PK)-resistant human α-syn aggregates, tissue slices were pre-stained with haematoxylin and digested with PK before the incubation with antibodies against α-syn (Neumann et al., 2004 (link)). Biotinylated horse anti-mouse IgG, biotinylated goat anti-rat IgG, and biotinylated goat anti-rabbit IgG (1:200; Vector Laboratories, USA) were used as secondary antibodies. Vectastain ABC reagent (Vector Laboratories, USA) and 3,3′-diaminobenzidine were applied to visualize the immunohistochemical binding sites. Stained sections were mounted onto gelatin-coated slides, dehydrated and coverslipped with Entellan.

Tenuigenin (molecular formula: C₃₀H₄₅ClO₆; average molecular weight: 537.14 kDa, chemical structure showed in Fig. 1) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (111572-200702) with a purity of 98.7%. LPS (Escherichia coli 0111:B4, L4391), MPTP (M0896), ATP (A2385), and MSU (U2875) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse IL-1β ELISA Kits were purchased from R&D Systems. ROS-specific fluorescent probe cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Invitrogen (Thermo Fisher Scientific Inc., USA). The antibodies used in this study are the following: rabbit monoclonal anti-NLRP3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit anti-caspase-1 (1:800, Santa Cruz Biotechnology, USA), goat anti-IL-1β (1:800, R&D Systems, Minneapolis, USA), mouse monoclonal anti-β-actin (1:5000, Sigma-Aldrich), mouse monoclonal anti-tyrosine hydroxylase (TH, 1:1000, Sigma-Aldrich), and rabbit polyclonal anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:500, Wako, Osaka, Japan). The reagents obtained from other sources are detailed throughout the following text.Fig. 1

Chemical structure of tenuigenin