Pathology Department, Mary Lyon Centre, Medical Research Council, Harwell, United Kingdom.
Mouse Models of Neurodegeneration, Mammalian Genetics Unit, Medical Research Council, Harwell, United Kingdom.
Curr Protoc. 2022 May;2(5):e428. doi: 10.1002/cpz1.428.
Histology is the study of the microscopic structure of tissues. This protocol permits the generation of frozen transverse sections of lumbar spinal cord regions L3 to L6. It enables counting of murine ventral horn lumbar motor neurons in a reproducible manner. Methods include spinal column dissection, hydraulic extrusion, and histological processing. The preparation for cryo-sectioning includes embedding lumbar spinal cord in optimal cutting temperature (OCT) medium. The correct orientation of the tissue is critical as calculating the amount of tissue to discard saved time overall. Specific details regarding section thickness and mounting are described. These requirements not only allow optimum coverage of specific regions but also ensure that no individual motor neuron was counted twice. The Nissl bodies of the motor neurons were stained using gallocyanin. The sections obtained are all of a comparable area and quality assurance is consistent. The specificity of the staining enables the scientist to identify and reliably quantify lumbar motor neurons. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Euthanasia of mouse and isolation of spinal cord Basic Protocol 2: Hydraulic extrusion of the spinal cord Basic Protocol 3: Identification of the lumbar region Basic Protocol 4: Embedding cord in OCT Basic Protocol 5: Collection of frozen sections onto slides Basic Protocol 6: Gallocyanin staining Basic Protocol 7: Motor neuron counting.
组织学是研究组织的微观结构的学科。本方案允许生成 L3 到 L6 腰椎脊髓区域的冷冻横向切片。它可以以可重复的方式计算小鼠腹角腰椎运动神经元的数量。方法包括脊柱解剖、液压挤压和组织学处理。用于冷冻切片准备的步骤包括将腰椎脊髓嵌入最佳切割温度（OCT）介质中。组织的正确定向至关重要，因为计算要丢弃的组织量可以节省总体时间。描述了有关切片厚度和安装的具体细节。这些要求不仅允许对特定区域进行最佳覆盖，还确保没有单个运动神经元被重复计数。使用硝酸银对运动神经元的尼氏体进行染色。获得的切片均具有可比的面积和一致的质量保证。染色的特异性使科学家能够识别并可靠地定量腰椎运动神经元。© 2022 作者。Wiley Periodicals LLC 出版的《当代协议》。基本方案 1：处死小鼠并分离脊髓基本方案 2：脊髓的液压挤压基本方案 3：腰椎区域的鉴定基本方案 4：将脊髓嵌入 OCT 基本方案 5：将冷冻切片收集到载玻片上基本方案 6：硝酸银染色基本方案 7：运动神经元计数。
