Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University.
Department of Neurology, State University of New York Upstate Medical University;
J Vis Exp. 2022 Jan 21(179). doi: 10.3791/63386.
The histologic analysis of brain and spinal cord specimens isolated from mice is common practice for the assessment of pathology in this model system. To maintain the morphology of these delicate tissues, it is routine to administer a chemical fixative such as paraformaldehyde via cannulation of the heart in anesthetized animals (transcardial perfusion). Transcardial perfusion of the mouse heart has traditionally relied on the use of peristaltic pumps or air pressure to deliver both the saline and fixative solutions necessary for this process. As an easily accessible alternative to these methods, this work demonstrates the use of a gravity-fed method of perfusate delivery that uses materials available in most hardware stores. To validate this new perfusion method, this work demonstrates all the subsequent steps necessary for the sensitive detection of phosphorylated α-synuclein in both the brain and spinal cord. Included in these steps are the dissection of the fixed brain and spinal cord tissues, rapid freezing/embedding and cryosectioning of the tissues, and immunofluorescent staining. As this method results in whole-body delivery of the fixative, it may also be used to prepare other non-neuronal tissues for histologic analysis.
从麻醉动物（心脏灌流）通过心脏套管插入术将化学固定剂（如多聚甲醛）施用于脑和脊髓标本以用于评估该模型系统中的病理学，这是一种常见的做法。为了保持这些精细组织的形态，通常需要使用蠕动泵或气压来输送进行此过程所需的盐水和固定剂溶液。作为对这些方法的一种易于获得的替代方法，这项工作展示了使用重力供给灌流液的方法，该方法使用大多数五金店都有的材料。为了验证这种新的灌注方法，这项工作展示了在大脑和脊髓中敏感检测磷酸化α-突触核蛋白所需的所有后续步骤。这些步骤包括固定的脑组织和脊髓组织的解剖、组织的快速冷冻/包埋和冷冻切片以及免疫荧光染色。由于该方法导致固定剂的全身输送，它也可以用于为组织学分析准备其他非神经元组织。
