β3 ar

β₁-AR, β₂-AR, β₃-AR (Santa Cruz Biotechnology, Santa Cruz, CA), G_αs, G_αi, and GAPDH (Cell Signaling Technology, Danvers, MA) protein expression was determined via Western Blot analysis. Thirty micrograms of left and right ventricular homogenates were resolved on SDS-PAGE gradient gels (4–15%, Tris-Glycine) and transferred to polyvinylidine difluoride membranes. Primary antibody incubation occurred at 4°C for a minimum of 16 hours. Blots were then washed with 0.1% TBST, followed by incubation with anti-HRP secondary antibodies (Cell Signaling) for a minimum of 1 hour at room temperature. Band intensities were visualized using chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, Rockford, IL, USA) and quantified using NIH ImageJ Data Acquisition Software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemical analyses were performed as previously described, with minor modifications [51 (link)]. The following primary antibodies and dilutions were used: OX-42 (1:1,000; Serotec, Oxford, UK), Iba1 (1:1,000; Wako Pure Chemical Industries, Osaka, Japan), CD11b (1:1,000; Serotec), DBH (1:1,000; Millipore, Bedford, MA, USA), Ki 67 (1:1,000: Abcam), Cleaved caspase 3 (1:1,000: Cell Signaling, MA, USA), β1-adrenergic receptor (AR) (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), β2-AR (1:1,000; Santa Cruz Biotechnology), and β3-AR (1:1,000; Santa Cruz Biotechnology). After PBSTx rinses, the sections were incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 60 min at 1:200 in 0.1 M PBST containing 1% BSA. Following rinsing, the sections were exposed to avidin–biotin horseradish peroxidase complex in 0.1 M PBST. Antigens were visualized through reaction with 0.05% 3,3′-diaminobenzidine tetrahydrochloride as a chromogen in PBS and 0.003% hydrogen peroxide for 5 min. The sections were mounted on gelatin-coated slides, dehydrated, and coverslipped with Multi-Mount (Matsunami Glass Ind., Ltd., Osaka, Japan).
For immunofluorescence, the following primary antibodies and dilutions were used: OX-42 (1:200), DBH (1:200), β1-AR (1:200), β2-AR (1:200), and β3-AR (1:200).

The paraffin sections were incubated in the rabbit polyclonal antibody β3-AR (1∶50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C. As a negative control, PBS was used in place of the primary antibody. Then TRITC-conjugated goat anti-rabbit IgG (1∶100 dilution, Jackson ImmunoResearch, West Grove, PA, USA) was used as the secondary antibody to detect the primary antigen-antibody reaction. The nuclei was stained by 4′-6-diamidino-2-phenylindole (DAPI) dye (1∶2000 dilution, Sigma, St Louis, MO, USA). Immunofluorescent labeling of the sections were observed with a fluorescence microscope (Nikon Eclipse 55i, Nikon, Tokyo, Japan). Quantification of the β3-AR fluorescence density was determined by IPP 6.0.