Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections
Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections
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DOI：
10.3791/53976
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发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Li, Huiliang
中科院分区：
文献类型：
--
作者：
Jolly, Sarah;Fudge, Alexander;Li, Huiliang
在不同类型的脑细胞（例如神经元、星形胶质细胞、少突胶质细胞、少突胶质前体细胞和小胶质细胞）中检测基因表达可能会因缺乏用于免疫染色的特异性一抗或二抗而受阻。在此我们描述一种方案，使用两种基因特异性探针进行双重荧光原位杂交，然后用一种针对由第三个基因编码的蛋白质的高特异性抗体进行免疫染色，从而在同一脑切片中检测三个不同基因的表达。天冬氨酸酶（ASPA）基因的突变可导致一种罕见的人类白质疾病——卡纳万病，该基因被认为在少突胶质细胞和小胶质细胞中表达，但不在星形胶质细胞和神经元中表达。然而，ASPA在大脑中的精确表达模式尚未确定。该方案使我们能够确定ASPA在一部分成熟少突胶质细胞中表达，并且它可普遍应用于广泛的基因表达模式研究。
Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.