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Author:
Date:
2014-09-20
[Abstract] In situ hybridization and immunostaining are common techniques for localizing gene expression, the mRNA and protein respectively, within tissues. Both techniques can be applied to tissue sections to achieve similar goals, but in some cases, it is necessary to use them together. For example, complement C1q is a secreted protein complex that can target the innate immune response during inflammation. Complement has been found to be elevated early and before severe neurodegeneration in several disease models. Thus, complement may serve as an important marker for disease progression and may contribute to the pathology under certain conditions. Since complement is a secreted complex, immunostaining for C1q does not necessarily reveal where compliment is produced. In situ ...
[摘要] 原位杂交和免疫染色是分别定位组织内基因表达，mRNA和蛋白质的常用技术。两种技术可以应用于组织切片以实现类似的目标，但是在一些情况下，有必要一起使用它们。例如，补体C1q是可以靶向炎症期间的先天免疫应答的分泌的蛋白复合物。已经发现补体在几种疾病模型中早期升高和严重神经变性之前升高。因此，补体可以作为疾病进展的重要标志物，并且可能有助于在某些条件下的病理学。由于补体是分泌的复合物，C1q的免疫染色不一定揭示产生补偿的位置。补体成分，C1q a，b或c mRNA的原位杂交是理想的标记组织中产生补体的细胞。原位杂交可以与细胞类型特异性免疫染色偶联以准确鉴定所涉及的细胞类型。蛋白质定位和mRNA定位一起可以揭示关于疾病组织内补体产生和补体靶细胞之间的关系的细节。在这里我们概述在同一组织切片上的组合原位杂交和免疫染色的步骤。这里概述的协议已设计用于检测小鼠脑神经元和小胶质细胞中的补体C1q。
这里提供了两种组合ISH/IH的方法。在第一个实施例中，C1q mRNA的原位杂交与使用Calbindin-D28K抗体的Purkinje神经元细胞体的荧光检测一起进行。在第二个实施例中，使用CD68抗体与小胶质细胞的3,3'-二氨基联苯胺（DAB）一起进行C1q ...
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