Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone.
Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone.
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DOI：
10.3791/59113
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发表时间：
2019-05
期刊：
影响因子：
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通讯作者：
Jingwen Yang;Haichun Pan;Y. Mishina
中科院分区：
文献类型：
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作者：
Jingwen Yang;Haichun Pan;Y. Mishina
组织免疫染色提供了对给定组织内感兴趣的蛋白质的高度特异性和可靠的检测。在这里，我们描述了一个完整的和简单的协议，以检测蛋白质的表达在颅面形态发生/发病机制，以小鼠颅面组织为例。该方案包括组织的制备和冷冻切片、间接免疫荧光、图像采集和定量。此外，还描述了一种用于免疫染色的未脱钙硬组织的制备和冷冻切片方法，以颅面组织和长骨为例。这些方法是确定颅面形态发生/发病过程中各种组织中蛋白质表达和形态/解剖学变化的关键。经适当修改后，它们也适用于其他组织。组织学知识和高质量的切片对于从实验结果中得出科学结论至关重要。这种方法的潜在局限性包括但不限于抗体的特异性和定量的困难，这也在这里讨论。
Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.