Pagliusi Marco, Brandão Arthur F, Zanetti Gabriel G, Bonet Ivan J M, Sartori Cesar R, Vieira André S
Department of Structural and Functional Biology, University of Campinas, Campinas, Brazil.
Bio Protoc. 2020 Dec 5;10(23):e3836. doi: 10.21769/BioProtoc.3836.
Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (., real-time PCR, Western blot, and RNA-seq).
显微切割技术对于专注于神经科学的解剖学和功能研究非常重要，在这些研究中，通常需要显微切割特定的脑区以进行分子或解剖学分析。之前已经描述了石蜡膜辅助显微切割（PAM），它涉及对安装在覆盖有石蜡膜的载玻片上的组织切片进行显微切割。在这项工作中，我们描述了使用PAM方法对啮齿动物伏隔核（NAc）进行显微切割的过程。（1）我们首先描述实施小鼠安乐死以及取出大脑的最佳方法。（2）接下来，我们描述如何准备用于接收脑切片的带有石蜡膜的载玻片。（3）随后，我们描述如何在低温恒温器中处理大脑，如何对齐半球以及如何确定NAc的前后界限。（4）我们还描述了如何对切片进行染色和脱水，这是促进显微切割并保存大分子的关键步骤。（5）在最后一步中，我们描述如何确定NAc的背腹和内外界限，最后描述如何对该区域进行手动显微切割。这是一种低成本技术，使研究人员能够特异性地显微切割任何脑区，从中可以提取完整的RNA和蛋白质以进行多种分子分析（如实时PCR、蛋白质印迹和RNA测序）。
