全山解剖和成年小鼠耳蜗的免疫
Summary
我们提出了一个方法，以分离科尔蒂的成年器官三个完整耳蜗匝（先端，中间，和碱）。我们还演示了一个程序，用荧光标记的抗体免疫染色。连同这些技术允许毛细胞，支持细胞，以及其他细胞类型中的耳蜗研究发现。
Abstract
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.
Introduction
内耳的螺旋形耳蜗，包含在颞骨内，容纳尔蒂，在哺乳动物听觉感官端器官的器官。耳蜗是tonotopically组织并通常分为顶，中间，和基底轮流对应于不同的频率范围具有高频率的声音的检测，在碱和低频检测在顶点1。毛细胞，Corti器的mechanosensory细胞，运行耳蜗，这大约是6毫米长的小鼠2,3的长度。这些细胞将声波转换，这是通过填充流体的膜迷路发送，进入通过中央听觉结构处理的神经信号的机械能。此处所描述的技术提供了制备柯蒂氏器的全样载耳蜗后钙化的方法完成（为样本范围从年龄一周成年）。我们还提出了immunosta的方法都进不去，整个安装耳蜗组织。人工耳蜗整个坐骑是所有毛细胞的可视化和周边配套细胞在自然空间布局的关键，并允许进行分析的三个维度与使用共焦显微镜。
博士。汉斯恩斯特龙和哈洛阿德斯最初描述整个支架耳蜗解剖法在1966年他们详述的技术迅速修复和解剖钙化耳蜗浸没在液体中从各种哺乳动物，保留柯蒂氏器的短完好分段微观分析4。不固定，钙化大鼠耳蜗的解剖也已在教学视频5所示 。博士。芭芭拉Bohne和加里·史密斯在华盛顿大学取得了一些重要的修改这个方法。在他们的耳蜗整装方法的版本，颞骨是脱钙，嵌入塑料，和五个半圈，十四分之一圈是dissec特德6,7。查尔斯·利伯曼博士，并在伊顿皮博迪实验室，麻省眼耳医院的同事，修改了此技术使塑料包埋是不需要8。该技术的进一步修改发生在践祚博士的实验室在圣裘德儿童研究医院9-12该通知这里介绍的清扫方法。我们使用了不同的策略来访问科尔蒂比Bohne和利伯曼，的器官，它允许完全根尖，中间，和基底匝隔离。因此，解剖组织较大且较不可能在解剖或免疫染色过程丢失或损坏。此外，目前的方法有利于从心尖尖端或基底钩，以确定的频率区域的距离的测量。
尽管许多实验室进行耳蜗组织免疫，目前还不清楚其中这些方法起源。其结果是有各种配方用于阻断buffeRS和抗体孵育缓冲液可能影响个体初级抗体的性能。这里，我们提出的一种方法使用本身是适用于听觉领域最常用的抗体荧光标记的抗体的免疫染色。
形状复杂的耳蜗，柯蒂氏器的精巧结构，和骨包套提供用于组织学和生物化学分析的一个挑战。多种技术的听证会现场，目前用于克服这些困难的特点和尔蒂，每种技术都有自己的优点和缺点的器官内可视化的细胞。这里介绍的协议允许成年小鼠耳蜗的整装解剖和，稍加修改，可潜在地用于检查耳蜗内的关键结构从各种在该领域中使用的其他模型生物。
Protocol
Representative Results
Discussion
有成功的整装解剖和免疫组化的几个关键步骤。但是前两种方法之一被执行，需要耳蜗组织的正确固定。我们建议使用甲醇免费，超纯水，EM级PFA。 PFA从粉末制成的可以有甲醇和不稳定的pH值从而降低免疫荧光的质量的痕迹。其他组也表明类似夹层是可能使用的固定剂不含有甲醛14-16。固定的长度也很重要，是抗体特异性的。一些抗体能够容忍的O / N固定，而另一些人不只是1小时正常?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by a grant from the Office of Naval Research (N000141310569). The Southern Illinois University School of Medicine Research Imaging Facility equipment was supported by the National Center for Research Resources-Health (S10RR027716).
Materials
| Standard pattern forceps | Fine Science Tools | 11000-12 | can be purchased from other vendors |
| 10.5 cm fine scissors | Fine Science Tools | 14060-11 | can be purchased from other vendors |
| 2 ml microcentrifuge tubes | MidSci | AVSS2000 | can be purchased from other vendors |
| 16% formaldehyde, methanol free, ultra pure, EM grade | Polysciences | 18814 | TOXIC –wear gloves and cannot be disposed of in the sink. Can be purchased from other vendors. |
| PBS pH 7.4 | Sigma | P3813-10PAK | can be purchased from other vendors |
| EDTA | Fisher | BP118-500 | can be purchased from other vendors |
| end-over-end tube rotator | Fisher | 05-450-127 | can be purchased from other vendors |
| 60 mm petri dish | Fisher | 50-202-037 | can be purchased from other vendors |
| Dow Corning Sylgard 184 silicone encapsulant kit | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | |
| activated charcoal | Fisher | AC134372500 | can be purchased from other vendors |
| stereo dissection microscope | Zeiss | Stemi 2000 | can be purchased from other vendors |
| Dumont #4 jeweler's forceps | Fine Science Tools | 11241-30 | |
| Dumont #5 jeweler's forceps | Fine Science Tools | 11251-20 | |
| 2.5 mm Vannas spring scissors | Fine Science Tools | 15001-08 | curved |
| 5 mm Vannas-Tubingen spring scissors | Fine Science Tools | 15003-08 | straight |
| 48 well plates | Fisher | 08-772-52 | can be purchased from other vendors |
| 8 well chamber slides | Fisher | 1256518 | can be purchased from other vendors |
| Triton X-100 | Sigma | X100-500 | can be purchased from other vendors |
| BSA, fraction V | Fisher | BP1605 | can be purchased from other vendors |
| NGS | Vector labs | S-1000 | can be purchased from other vendors |
| NHS | Vector labs | S-2000 | can be purchased from other vendors |
| 3D rotator | Midsci | R3D-710 | can be purchased from other vendors |
| Western blot incubation box XL | Licor | 929-97401 | |
| Hoechst 33342 | Life Technologies | H3570 | can be purchased from other vendors |
| Prolong gold antifade mounting media | Life Technologies | P36930 | can be purchased from other vendors,but mounting medias vary in their ability to protect against photobleaching |
| Superfrost Plus Slides | Fisher | 12-550-15 | can be purchased from other vendors |
| coverslips 22 x 22 x 1 | Fisher | 12-548-B | can be purchased from other vendors |
| clear nail polish | Local drug store | can be purchased from other vendors | |
| cardboard slide folder | Fisher | 12-587-10 | can be purchased from other vendors |
| plastic slide box | Fisher | 03-448-10 | can be purchased from other vendors |
Riferimenti
- Robles, L., Ruggero, M. A. Mechanics of the mammalian cochlea. Physiol Rev. 81 (3), 1305-1352 (2001).
- Ehret, G., Frankenreiter, M. Quantitattive analysis of cochlear structures in the house mouse in relation to mechanisms of acoustical information processing. J Comp Physiol. 122, 65-85 (1977).
- Ou, H. C., Bohne, B. A., Harding, G. W. Noise damage in the C57BL/CBA mouse cochlea. Hear Res. 145 (1-2), 111-122 (2000).
- Engstrom, H., Ades, H. W., Andersson, A. . Structural pattern of the organ of Corti. A systematic mapping of sensory cells and neural elements. , (1966).
- Grant, L., Yi, E., Goutman, J. D., Glowatzki, E. Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse. J Vis Exp. (48), e2442 (2011).
- Bohne, B. A., Harding, G. W. Processing and analyzing the mouse temporal bone to identify gross, cellular and subcellular pathology. Hear Res. 109 (1-2), 34-45 (1997).
- Bohne, B. A., Harding, G. W., Ou, H. C., Willott, J. . Handbook of Mouse Auditory Research. , 171-187 (2001).
- Weber, T., et al. Rapid cell-cycle reentry and cell death after acute inactivation of the retinoblastoma gene product in postnatal cochlear hair cells. Proc Natl Acad Sci U S A. 105 (2), 781-785 (2008).
- Yu, Y., et al. In vivo proliferation of postmitotic cochlear supporting cells by acute ablation of the retinoblastoma protein in neonatal mice. J Neurosci. 30 (17), 5927-5936 (2010).
- Steigelman, K. A., et al. Polycystin-1 Is Required for Stereocilia Structure But Not for Mechanotransduction in Inner Ear Hair Cells. J Neurosci. 31 (34), 12241-12250 (2011).
- Liu, Z., et al. Age-dependent in vivo conversion of mouse cochlear pillar and Deiters’ cells to immature hair cells by Atoh1 ectopic expression. J Neurosci. 32 (19), 6600-6610 (2012).
- Parker, M., Brugeaud, A., Edge, A. S. Primary culture and plasmid electroporation of the murine organ of Corti. J Vis Exp. (36), e1685 (2010).
- Burns, J. C., On, D., Baker, W., Collado, M. S., Corwin, J. T. Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and striolar growth zones. J Assoc Res Otolaryngol. 13 (5), 609-627 (2012).
- Collado, M. S., et al. The postnatal accumulation of junctional E-cadherin is inversely correlated with the capacity for supporting cells to convert directly into sensory hair cells in mammalian balance organs. J Neurosci. 31 (33), 11855-11866 (2011).
- Meyers, J. R., Corwin, J. T. Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium. J Neurosci. 27 (16), 4313-4325 (2007).
- Gratton, M. A., Rao, V. H., Meehan, D. T., Askew, C., Cosgrove, D. Matrix metalloproteinase dysregulation in the stria vascularis of mice with Alport syndrome: implications for capillary basement membrane pathology. Am J Pathol. 166 (5), 1465-1474 (2005).
