Paletzki Ronald F, Gerfen Charles R
National Institute of Mental Health, Section on Neuroanatomy, Bethesda, Maryland.
Curr Protoc Neurosci. 2019 Dec;90(1):e84. doi: 10.1002/cpns.84.
This unit covers some basic procedures that are common to a wide range of neuroanatomical protocols for brain tissue. Procedures are provided for preparation of unfixed fresh brain tissue as well as for perfusion fixation of animals to obtain fixed neural tissue. A variety of methods for sectioning are described, including frozen sectioning using a cryostat or microtome and sectioning with a vibratome. The choice of sectioning method depends on how the brain has been prepared and what histochemical method is to be used. A fluorescent immunohistochemical method to localize endogenous molecules as well as induced markers such as green fluorescent protein and red fluorescent protein is also provided. Additionally, three post-sectioning procedures are described: defatting of slide-mounted sections, fluorescent Nissl staining, and thionin staining of sections. Finally, support protocols are provided, describing a method for maintaining the correct order of cut tissue, whether rostral to caudal or lateral to medial; a procedure for subbing slides with gelatin, which is necessary in some protocols in order for sections to adhere to slides; and preparation of custom 3D-printed 10- or 20-well tissue plates and trays for subsequent immunostaining. Published 2019. U.S. Government. Basic Protocol 1: Preparation of unfixed fresh-frozen brain tissue Basic Protocol 2: Perfusion fixation Basic Protocol 3: Cryostat sectioning of frozen brain tissue Basic Protocol 4: Sliding-microtome sectioning of fixed brain tissue Basic Protocol 5: Vibratome and Compresstome sectioning Support Protocol 1: Tissue collection in a 1-in-10 series Support Protocol 2: Preparation of gelatin-subbed microscope slides Support Protocol 3: Custom 3D-printed 10- and 20-well tissue plates Basic Protocol 6: Post-sectioning procedures I: Fluorescent immunohistochemical localization Basic Protocol 7: Post-sectioning procedures II: Defatting Basic Protocol 8: Post-sectioning procedures III: Nissl staining Basic Protocol 9: Post-sectioning procedures IV: Thionin staining.
本单元涵盖了一系列广泛的脑组织神经解剖学实验方案中常见的一些基本步骤。提供了未固定新鲜脑组织的制备方法以及对动物进行灌注固定以获取固定神经组织的方法。描述了多种切片方法，包括使用低温恒温器或切片机进行冷冻切片以及使用振动切片机进行切片。切片方法的选择取决于脑组织的制备方式以及要使用的组织化学方法。还提供了一种荧光免疫组织化学方法，用于定位内源性分子以及诱导标记物，如绿色荧光蛋白和红色荧光蛋白。此外，还描述了三种切片后程序：载玻片上切片的脱脂、荧光尼氏染色和切片的硫堇染色。最后，提供了辅助实验方案，描述了一种保持切割组织正确顺序的方法，无论是从吻侧到尾侧还是从外侧到内侧；一种用明胶包被载玻片的程序，在某些实验方案中这是使切片粘附在载玻片上所必需的；以及制备定制的3D打印10孔或20孔组织板和托盘用于后续免疫染色。2019年发布。美国政府。基本方案1：未固定新鲜冷冻脑组织的制备 基本方案2：灌注固定 基本方案3：冷冻脑组织的低温恒温器切片 基本方案4：固定脑组织的滑动切片机切片 基本方案5：振动切片机和压缩切片机切片 辅助方案1：1:10系列组织采集 辅助方案2：明胶包被显微镜载玻片的制备 辅助方案3：定制的3D打印10孔和20孔组织板 基本方案6：切片后程序I：荧光免疫组织化学定位 基本方案7：切片后程序II：脱脂 基本方案8：切片后程序III：尼氏染色 基本方案9：切片后程序IV：硫堇染色。
