StereoMate: 3D Stereological Automated Analysis of Biological Structures

Abstract

Tissue clearing methods offer great promise to understand tissue organisation, but also present serious technical challenges. Generating high quality tissue labelling, developing tools for demonstrably reliable and accurate extraction, and eliminating baises through stereological technique, will establish a high standard for 3D quantitative data from cleared tissue. These challenges are met with StereoMate, an open-source image analysis framework for immunofluorescent labelling in cleared tissue. The platform facilitates the development of image segmentation protocols with rigorous validation, and extraction of object-level data in an automated and stereological manner. Mouse dorsal root ganglion neurones were assessed to validate this platform, which revealed a profound loss and shift in neurone size, and loss of axonal input and synaptic terminations within the spinal dorsal horn following their injury. In conclusion, the StereoMate platform provides a general-purpose automated stereological analysis platform to generate rich and unbiased object-level datasets from immunofluorescent data.

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…This protocol and associated tools can be used to isolate ganglia from mice aged P5 onwards. The resulting DRG can be subsequently used for analyses of RNA and protein, immunohistochemically investigated as sections [15] and when cleared [14], or cultured as explants and dissociated primary neurons [19,25] for the in vitro study of live cellular processes [22]. Moreover, assessment of sensory neuron biology can be simultaneously compared and contrasted with that of the motor nervous system [23,[26][27][28] for detailed peripheral nerve characterisation.…”

“…neurotrophins) [7]. DRG have been isolated from several different mammalian species [8][9][10][11][12], and even human organ donors [13], and studied to better understand sensory neuron biology, including responses to nerve injury and disease [14][15][16], with experiments on mouse ganglia being most common. Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), ve or six lumbar (L1 to L5/L6) and four sacral (S1 to S4).…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…By following this step-by-step video-assisted protocol, you will be able to swiftly learn how to dissect mouse sensory ganglia from defined cervical, thoracic, and lumbar levels of the spinal column (> 50 DRG/animal). The resulting DRG can be subsequently used for analyses of RNA and protein, immunohistochemically investigated as sections [15] and when cleared [14], or cultured as explants and dissociated primary neurons [19,25] for the in vitro study of live cellular processes [22].…”

“…neurotrophins) [7]. DRG have been isolated from several different mammalian species [8][9][10][11][12], and even human organ donors [13], and studied to 3 better understand sensory neuron biology, including responses to nerve injury and disease [14][15][16], with experiments on mouse ganglia being most common. Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), five or six lumbar (L1 to L5/6) and four sacral (S1 to S4).…”

A video protocol for rapid dissection of mouse dorsal root ganglia from defined spinal levels

“…neurotrophins) [7]. DRG have been isolated from several different mammalian species [8][9][10][11][12], and even human organ donors [13], and studied to better understand sensory neuron biology, including responses to nerve injury and disease [14][15][16], with experiments on mouse ganglia being most common. Mice have 30 or 31 DRG pairs, depending on genetic background [17][18][19]: eight cervical (C1 to C8), 13 thoracic (T1 to T13), five or six lumbar (L1 to L5/L6) and four sacral (S1 to S4).…”