Frozen mouse brain sections were prepared according to previous protocol [42 (link)]. Mouse brains were dissected and fixed in 4% paraformaldehyde (Sigma-Aldrich, #158127) overnight at 4°C. Primary cultured raphe neurons were fixed in 4% paraformaldehyde for 30 min at 37°C. Brain slices or cultured raphe neurons were treated with blocking buffer (5% goat or donkey serum (Sigma-Aldrich), 0.3% Triton X-100 (Fisher Scientific) in PBS, pH 7.4) and probed with primary antibodies accordingly: rabbit-anti-serotonin (Sigma-Aldrich, #S5545, 1:400), rabbit-anti-serotonin transporter (abcam, #ab25358, 1:400), mouse-anti-ATP5B (Santa Cruz, #sc-55597, 1:500). After overnight incubation with primary antibodies, the brain slices or neurons were probed with proper cross-adsorbed secondary antibodies conjugated to Alexa Fluor 488, Alexa Fluor 594, or Alexa Fluor 647 (Thermo Fisher Scientific, 1:500). Cell nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Images were collected on a Nikon confocal microscope. Serotonin intensity, serotonergic fiber density, and mitochondrial volume and density were analyzed using Nikon-Elements Advanced Research software. Mitochondrial density was calculated as number of mitochondria per μm serotonergic fiber.