Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea
Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea
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DOI：
10.3791/53561
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发表时间：
2016-01-01
影响因子：
1.2
通讯作者：
Cox, Brandon C.
中科院分区：
文献类型：
--
作者：
Montgomery, Scott C.;Cox, Brandon C.
柯蒂氏器位于内耳耳蜗中，包含机械感觉毛细胞和周围的支持细胞，这些细胞以螺旋形状组织，并具有用于声音检测的音调梯度。小鼠耳蜗长约 6 毫米，通常分为三圈（顶端、中部和基部）进行分析。为了研究细胞损失、细胞分裂或镶嵌基因表达，耳蜗的整体或表面制备是有用的。当与免疫染色和共聚焦显微镜相结合时，这种解剖方法可以对 Corti 器官内的所有细胞进行可视化，以对 z 轴不同平面上的细胞进行成像。还可以从这些 z 堆栈图像中获得多个光学横截面。此外，尽管需要不同的固定方法，但整体解剖方法可用于扫描电子显微镜。在这里，我们提出了一种将柯蒂氏器分离为三个完整的耳蜗转（顶端、中部和基部）的方法。该方法可用于从一周龄到成年的小鼠，与用于耳蜗钙化不完全的新生儿样本的技术不同。稍作修改的版本可用于解剖大鼠耳蜗。我们还演示了使用荧光标记抗体进行免疫染色的程序。
The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.