Cm1100

Briefly, C57BL/6 male mice (5–8 weeks old) were intravenously injected with 5 × 10⁵ B16F10 cells to establish a lung metastasis model [29 (link)]. On the next day, the mice were intraperitoneally injected with PBS (vehicle control) or 2-DG (50 mg/kg) in PBS for 5 days. The mice were sacrificed by cervical dislocation on day 14 after tumor cell injection. The lungs and cardiac blood were obtained from these mice for ELISA and western blotting assays. To evaluate effects of 2-DG on tumor progression and intratumoral IL-1β levels, C57BL/6 mice (5–8 weeks old) were subcutaneously injected with 5 × 10⁵ B16F10 cells. PBS or 2-DG (50 mg/kg) in PBS was subcutaneously injected into the tumoral regions on days 7–14, and the tumor tissues were extracted on day 14. Following tumor volume measurements using the Ellipsoid formula (a × b × c × π × 4/3), B16F10-derived tumor tissues were frozen in FSC 22 (Surgipath, Richmond, IL, USA). Next, 5-μm cryosections were cut using a Leica CM1100 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) with microtome blades (Leica 818, Leica Microsystems GmbH, Wetzlar, Germany). Then, the tumor tissues were mounted on poly-L-lysine-coated glass slides (Muto Pure Chemicals, Tokyo, Japan). Tumor tissues were stained with anti-IL-1β, IB-4, and DAPI and analyzed by confocal microscopy.

Dihydroethidium (DHE; Molecular Probes, Eugene, OR, USA) was used to evaluate the amount of oxidant formation [33 (link)]. Frozen sections of aortic rings and carotid arteries were sectioned at 10 µm thickness by cryostat microtome (Leica CM1100; Leica Instruments, Germany) and incubated in Krebs solution containing 5 µM DHE for 10 min at 37 °C. Samples were examined with a confocal microscope (FV1000; Olympus, Tokyo, Japan) at an excitation/emission of 488/605 nm. Fluorescence intensity was quantified by Fluoview version 1.5 (FV10-ASW1.5; Olympus, Tokyo, Japan).

Histological confirmation of cannula placements in the BNST for drug microinjection was performed after the behavioral experiments. Mice were transcardially perfused with ice-cold 0.1 M phosphate-buffered saline (PBS) for 15 min, followed by filtered ice-cold 4% paraformaldehyde (PFA) in 0.1 M PBS for 15 min. The brains were carefully removed and further post-fixed in filtered 4% PFA for at least 24 h, then 30% sucrose at 4 °C for at least 48 h. Afterward, 40-µm-thick sections were cut from the frozen brain block, using a cryostat at −15 °C (Leica CM1100; Leica Biosystems, Nußoch, Germany), and mounted on poly-L-lysine-coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan; Cat. # S7441). After washing with PBS for 5 min, the sections were incubated with Mayer's Hematoxylin Solution (Wako Pure Chemical Industries Ltd.; Cat. # 131–09665) at room temperature (25 ± 2 °C) for 5 min. The hematoxylin-stained sections were washed with 50 °C water for 5 min. Images were captured using an 8-LED USB Digital Microscope Endoscope Magnifier Camera (FB-CMXW02; Koolertron, Shenzhen, China) equipped with Micro Capture Pro software (Celestron, LLC, Torrance, CA, USA). Probe placements were verified by adapting the Paxinos mouse brain atlas [34] , mainly for the dorsal and ventral BNST (Figure 1B and C). Mice with misplaced cannula were excluded from statistical analysis.