Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.
Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.
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分离小鼠眼周组织，用于眼外肌及其卫星细胞的组织学和免疫染色分析。
DOI：
10.1007/978-1-4939-3810-0_9
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Yablonka-Reuveni,Zipora
中科院分区：
文献类型：
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作者：
Stuelsatz,Pascal;Yablonka-Reuveni,Zipora
眼外肌（EOMs）由一组高度专业化的骨骼肌组成，控制眼球运动。尽管EOMs的一些独特特征（包括它们在杜氏肌营养不良症中的作用）引起了人们的持续关注，但对这些难以触及的肌肉的了解仍然有限。本章的目的是为小鼠EOMs的分离和组织学分析提供详细的方法。我们首先简要介绍眼外主肌和副肌的基本解剖和已建立的命名法。然后，我们提供了详细的描述与一步一步的图像，我们的程序分离（随后冷冻切片）EOMs，同时保持其原始结构组织的完整性。接下来，我们提出了我们经常使用的几种有用的组织学方案，包括：(1)使用myodcrex R26mTmGreporter小鼠来突出眼周组织的一般组织，该小鼠可以很好地区分肌肉（myodcrex驱动的GFP+）和非肌源性成分（番茄+）；(2) H&E染色分析，例如检测EOMs中不存在的患肢和膈肌中肌营养不良蛋白缺失表型的病理特征；(3)采用Pax7/层粘连蛋白双免疫染色法检测肌纤维基底膜下天然位置的肌原性祖细胞（即卫星细胞）。这里描述的EOM组织采集程序也可以适用于分离和研究卫星细胞和其他细胞类型。总的来说，本章中描述的方法应该为研究人员进入EOM研究领域提供必要的工具，并有助于更好地理解这一高度专业化的肌肉群及其复杂的微观解剖。
The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoDCre× R26mTmGreporter mouse that elegantly distinguishes muscle (MyoDCre-driven GFP+) from the non-myogenic constituents (Tomato+); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.