Pipeline for fluorescent imaging and volumetric analysis of neurons in cleared mouse spinal cords

• Published in: STAR protocols 2022-12, Vol.3 (4), p.101759-101759, Article 101759
• Main Authors: Yu, Hao, Li, Qiang, Sandoval, Alfredo, Gibbs, Holly C., English, Amber, Dunn, Tiffany, Moth, John, Elahi, Hajira, Chen, Bo
• Format: Article
• Language: English
• Subjects: Animals; Cell Biology; Image Processing, Computer-Assisted; Mice; Microscopy; Microscopy, Fluorescence - methods; Molecular/Chemical Probes; Neurons; Neuroscience; Protocol; Spinal Cord
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101759

Precisely measuring the number and somatic volume of neurons in the central nervous system at single-cell resolution is technically challenging. Here, we combine multiple techniques to address this challenge in optically cleared mouse spinal cords. We describe in vivo neuron labeling approaches, tissue-clearing technology, light sheet fluorescence microscopy, and machine learning-guided imaging analysis. This combination provides a precise determination of the cell number and somatic volume of any neuron population in the spinal cords. [Display omitted] •Retrograde labeling of a specific group of motor neurons in the mouse spinal cord•CUBIC tissue clearing of spinal cord samples for high-resolution volumetric imaging•Light sheet fluorescence microscopy-based imaging and machine learning-based analysis•Precise determination of the number and somatic volume of neurons in the spinal cords Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Precisely measuring the number and somatic volume of neurons in the central nervous system at single-cell resolution is technically challenging. Here, we combine multiple techniques to address this challenge in optically cleared mouse spinal cords. We describe in vivo neuron labeling approaches, tissue-clearing technology, light sheet fluorescence microscopy, and machine learning-guided imaging analysis. This combination provides a precise determination of the cell number and somatic volume of any neuron population in the spinal cords.