Wager-Miller Jim, Murphy Green Michelle, Shafique Hana, Mackie Ken
Department of Psychological and Brain Sciences, Gill Center for Biomolecular Research, Indiana University;
Department of Psychological and Brain Sciences, Gill Center for Biomolecular Research, Indiana University.
J Vis Exp. 2020 Apr 23(158). doi: 10.3791/60474.
As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5-1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 °C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.
随着我们对神经生物学理解的不断深入，分子分析常常在诸如内侧前额叶皮质（mPFC）或伏隔核等小的脑区进行。这项工作面临的挑战是在保留待检测微环境的同时，精确解剖出正确的区域。在本文中，我们描述了一种利用大多数实验室都容易获得的资源的简单、低成本方法。该方法通过在整个过程中保持组织冷冻来保存核酸和蛋白质。使用脑基质将大脑切成0.5 - 1.0毫米的切片，并排列在冷冻的玻璃板上。将每个切片内的地标与参考图谱（如艾伦小鼠脑图谱）进行比较，然后使用冷手术刀或活检打孔器解剖出相应区域。随后将组织储存在-80°C直至使用。通过这个过程，已经成功地使用qRT-PCR和蛋白质免疫印迹分析对大鼠和小鼠的mPFC、伏隔核、背侧和腹侧海马以及其他区域进行了分析。该方法仅限于可以通过清晰地标识别的脑区。
