该做什么和不该做什么的肌肉冰冻切片的准备组织学分析
Summary
Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.
Abstract
肌肉活检组织学评价曾担任在神经肌肉疾病的病理发展过程的理解不可或缺的工具。然而，为了这样做，适当的照料需要切除并保存组织，以达到最佳的染色时，必须考虑。组织保存的一种方法包括固定在甲醛组织，然后用石蜡包埋它们。此方法保留形态良好，并且允许长期储存于RT但是麻烦的，并且需要处理的有毒化学品。此外，甲醛固定导致抗原的交联，这需要抗原修复协议有效免疫染色。与此相反，冷冻切片不需要固定，如此保留生物抗原的构象。此方法还提供了一个明显的优势在周围时间快转，使得它在情况需要像intraopera快速组织学评估特别有用略去手术活检。这里，我们描述制备肌肉活检用于可视化具有不同的组织学和免疫染色的最有效的方法。
Introduction
检查肌肉组织学中起着不同类型神经肌肉疾病1-4中的认识至关重要的作用。当与其他技术相结合，它使研究人员能够更好地了解病理的表现和进展的，也可以是必需评估治疗干预5-10的功效。然而，要准确，组织需要以适当的方式被保留以便切除之前立即保持生理状态。这种去除，保存，切片，和染色过程中需要非常小心。
组织可以以两种不同的方式进行光学显微镜研究来制备。第一种方法，福尔马林固定的石蜡包埋（FFPE）的部分，需要组织到被嵌入石蜡11,12之前被固定在10％的天然缓冲甲醛。固定步骤，有助于更好地保存形态，而且交联内源性PRoteins因而需要复杂的抗原修复程序用于免疫染色和消除酶染色对这些部分12的可能性。冰冻切片，另一方面，不需要进行预固定或处理;因此，蛋白质能保留原生生物构13,14。此外，冷冻切片也允许快速的周转时间，这有时是必要的，例如在肉瘤和其他外科活组织检查15的术中诊断。但是，如果没有正确完成，该方法是容易冻结损坏，它引入了改变肌肉结构使得部分不适宜用于组织学检查。本文将概述准备肌肉活检，以达到正确检查最佳染色最有效的方法。
Protocol
Representative Results
Discussion
组织学和免疫组化已被用作主要的工具在理解表现和病理的进展在各种神经肌肉疾病。经典地，肌肉活检第一固定在甲醛，然后用石蜡包埋蜡。虽然甲醛固定保持组织结构，并允许组织存储和运输，在RT，这也灭活酶和蛋白质交联迫使进一步措施，以实现精确的免疫染色。这个问题与使用冰冻切片消除，但这种方法提出了获得最佳的染色效果和准确的诠释14,15独特的技术挑战。
<p class="j…Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).
Materials
| Cryostat | Leica CM 1850 | Leica Microsystems Inc. | Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 Richard Allen Scientific |
| Microscope | Nikon Eclipse 50i | Nikon Instruments Inc. | 1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. Phone: +1-631-547-8500 Olympus |
| Image analysis software | Nikon Imaging Software Basic Research | Nikon Instruments Inc. | 1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. Phone: +1-631-547-8500 Image J, Metamorph |
| Isoflurane | 14043-220-05 | JD Medical Dist. Co., Inc. | JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029 |
| OCT | 4583 | Sakura Inc. | Sakrua Fintek, USA, Torrence, CA 90501, USA Leica Microsystem |
| Freeze It | 23-022524 | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| Disposable tissue mold | 22-363-554 | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| Colorfrost plus slides | 9991001 | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| Acetone | 650501-4L | Sigma-Aldrich | 3050 Spruce Street, St.Louis MO, 63103 VWR Scientific |
| Ethyl alcohol | HC-1300-1GL | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| 2-methyl butane | M 32631-SL | Sigma-Aldrich | 3050 Spruce Street, St.Louis MO, 63103 VWR Scientific |
| Xylene | HC-7001GA | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| Cytoseal 280 | 8311-4 | Thermofisher | 790 Memorial Dr, Cambridge, MA 02139 VWR Scientific |
| Hematoxylin Gill #3 | CS402-1D | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
| Eosin | 6766007 | Thermofisher | 790 Memorial Dr, Cambridge, MA, USA, 02139 VWR Scientific |
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