Mice were first deeply anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg), then transcardially perfused with PBS, followed by ice-cold 4% paraformaldehyde (PFA). After removing the cerebral cortex, the brainstem was left in the skull and postfixed in the same fixative overnight at 4°C. Parasagittal or coronal sections, 40–50 μm thick, of the CN were cut with a vibratome (Leica 1000S).
Free floating sections were collected, permeabilized, blocked with 3% BSA, and incubated with primary anti-GFP antibody (rabbit, Abcam catalog #ab290, 1:500) overnight on a shaker at 4°C to preserve the fluorescent signal of CeFP of the hVOS probe in TRAP-labeled neurons. In some experiments, sections were double stained with anti-tubulin β-III antibody (TUBB3, mouse, BioLegend catalog #801202, 1:500), as a neuronal marker. Sections were then incubated with fluorescent-dye conjugated secondary antibodies for 2 h (Alexa 568 anti-rabbit, Abcam catalog #ab175692, 1:500; Alexa 488 anti-mouse, 1:500, ThermoFisher Scientific catalog #A21202) and counterstained with DAPI for 10 min at room temperature. Images were acquired with a confocal microscope (Nikon A1RS) and processed with ImageJ.
Free floating sections were collected, permeabilized, blocked with 3% BSA, and incubated with primary anti-GFP antibody (rabbit, Abcam catalog #ab290, 1:500) overnight on a shaker at 4°C to preserve the fluorescent signal of CeFP of the hVOS probe in TRAP-labeled neurons. In some experiments, sections were double stained with anti-tubulin β-III antibody (TUBB3, mouse, BioLegend catalog #801202, 1:500), as a neuronal marker. Sections were then incubated with fluorescent-dye conjugated secondary antibodies for 2 h (Alexa 568 anti-rabbit, Abcam catalog #ab175692, 1:500; Alexa 488 anti-mouse, 1:500, ThermoFisher Scientific catalog #A21202) and counterstained with DAPI for 10 min at room temperature. Images were acquired with a confocal microscope (Nikon A1RS) and processed with ImageJ.
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