Imaging of 50 µm sections from perfusion-fixed brain was conducted on a Nikon Ti2 Eclipse epifluorescence microscope or on a Leica SP8 scanning confocal microscope. To confirm the specificity of V5 expression in mDA neurons, tile scan epifluorescence images of the entire VM were collected at 2–3 z-planes per section. V5-positive neurons were first identified using only the V5 channel and their somas were segmented as ROIs. Each V5-positive neuronal ROI was subsequently scored for tdTomato and TH expression. Neurons within both the SNc and ventral tegmental area were quantified. High-resolution (×60/1.4 NA) confocal images of striatal sections confirmed that V5 (APEX2), tdTomato, and TH were localized exclusively within dopaminergic axons. 300-µm-thick brain slices were imaged only by confocal microscopy. To assess biotin labeling throughout the slice, Z-stacks spanning the entire slice depth were acquired at 4.18 µm intervals using a ×20/0.4 NA objective.
Ti2 eclipse epifluorescence microscope
Manufactured by Leica
The Ti2 Eclipse epifluorescence microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It features a sophisticated optical system, a powerful illumination source, and a versatile platform for accommodating a range of sample types and experimental configurations.
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2 protocols using ti2 eclipse epifluorescence microscope
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Imaging Dopaminergic Neuron Expression
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Visualizing Dopaminergic Neuron Markers
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Imaging of 50 µm sections from perfusion-fixed brain was conducted on a Nikon Ti2 Eclipse epifluorescence microscope or on a Leica SP8 scanning confocal microscope. To confirm the specificity of V5 expression in DA neurons, tile scan epifluorescence images of the entire ventral midbrain were collected at 2-3 z-planes per section. V5-positive neurons were first identified using only the V5 channel and their somas were segmented as ROIs. Each V5-positive neuronal ROI was subsequently scored for tdTomato and TH expression. Neurons within both the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) were quantified. High resolution (60x/1.4 NA) confocal images of striatal sections confirmed that V5 (APEX2), tdTomato, and TH were localized exclusively within dopaminergic axons. 300 µm thick brain slices were imaged only by confocal microscopy. To assess biotin labeling throughout the slice, Z-stacks spanning the entire slice depth were acquired at 4.18 µm intervals using a 20x / 0.4 NA objective.
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