Double pre-embedding immunogold labeling for observation at transmission electron microscopy (TEM) was performed in the VTA (50-μm-thick) of TH-eGFP fed ad libitum mice fixed with 3% paraformaldehyde/0.5–1% glutaraldehyde (vol/vol) in PB. The TH-eGFP neurons were selected by observation under appropriate epifluorescence microscopy by being easily recognizable at 488 nm excitation wavelength. The selected sections were incubated free-floating overnight at 4°C with the primary antibodies (rabbit anti-CB1R antibody, anti-C terminus 461–472, Abcam; and goat anti- OX1R, Santa Cruz), all diluted 1:100 in donkey serum blocking solution with 0.02% saponin. Subsequently, the sections were incubated in a mixture of 6 nm (for CB1R) and 10 nm (for OX1R) gold-conjugated secondary antibodies (Aurion), diluted 1:30 in donkey serum blocking solution with 0.02% saponin. Sections were treated with 0.5% OsO4 in PB for 30 min at 4°C, dehydrated in an ascending series of ethanol and propylene oxide, and embedded in TAAB 812 resin (TAAB). Ultrathin (50 nm thickness) sections were cut by vibratome (Leica), collected on Formvar-coated, single- or multiple-slot (50-mesh) grids, and stained with 0.65% lead citrate. Electron micrographs were taken with the TEM microscope (FEI Tecnai G2 Spirit TWIN). The TEM observation was limited to series sectioned up to 0.6–0.8 μm depth from the external surface of pre-embedded immunolabeled tissue. Additional sections were processed in parallel as controls of reaction by omitting both or one of the primary antibodies from the mixture. No labeling was detected in the control material.

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