Multi mount

HMab-2 antibody was used for immunohistochemistry. The tumor samples were fixed with 10% formalin and embedded with paraffin. Sections (5-μm thick) were prepared using a microtome (RM2125RT, Leica, Wetzlar, Germany). After deparaffinization and hydration, the sections were incubated in retrieval solution, Tris-EDTA buffer pH 9.0, for 30 min at 100 °C in an electric pot and then blocked with 1.5% normal goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS containing 0.05% Tween-20, at room temperature for 1 h, and were incubated with HMab-2, diluted to 1 μg ml^–1, overnight at 4 °C. The second labeled polymer from the EnVision HRP kit (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was applied and the sections were incubated for 30 min. The substrate-chromogen solution from the DAB Substrate Kit (Vector Laboratories) was applied for 10 min. After washing, the sections were counterstained with hematoxylin and mounted in multi-Mount (Matsunami Glass Ind., Kishiwada, Japan).

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).