Kikuchi T, Mori S, Nishikawa S I, Yokota Y
Department of Molecular Genetics, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan.
J Immunol Methods. 2000 Jun 23;240(1-2):15-22. doi: 10.1016/s0022-1759(00)00163-0.
We describe a method to identify cells expressing mRNA of interest in the developing digestive tract by whole mount in situ hybridization with digoxigenin-labeled RNA probes. In preparing samples, serosal tissue surrounding the intestine was removed. Enzymatic reactions and probe concentrations were optimized. Furthermore, polyvinyl alcohol was included in the reaction mixture for the color development of alkaline phosphatase conjugated to the antibody against digoxigenin. These modifications improved the sensitivity and enabled us to identity cells that express mRNA in embryonic intestine. Using the antisense probe for VCAM-1, the protein product of which is an immunohistochemical marker of the Peyer's patch in the embryonic intestine, cells expressing mRNA were identified as spot-like clusters in Peyer's patches, confirming the validity of the method. With this method, mRNAs of both lymphotoxins alpha and beta, key molecules for peripheral lymphoid organ development, were found to be confined to the Peyer's patch in the developing intestine. Whole mount in situ hybridization analysis is a useful tool for exploring spatio-temporal expression profiles of mRNA in the developing immune organs.
我们描述了一种通过用地高辛标记的RNA探针进行全组织原位杂交，来鉴定发育中消化道内表达感兴趣mRNA的细胞的方法。在制备样本时，去除了肠道周围的浆膜组织。对酶促反应和探针浓度进行了优化。此外，在反应混合物中加入聚乙烯醇，用于与抗地高辛抗体偶联的碱性磷酸酶的显色。这些改进提高了灵敏度，使我们能够鉴定出胚胎肠道中表达mRNA的细胞。使用针对VCAM-1的反义探针，其蛋白质产物是胚胎肠道中派尔集合淋巴结的免疫组织化学标志物，表达mRNA的细胞在派尔集合淋巴结中被鉴定为点状簇，证实了该方法的有效性。通过这种方法，发现外周淋巴器官发育的关键分子淋巴毒素α和β的mRNA都局限于发育中肠道的派尔集合淋巴结。全组织原位杂交分析是探索发育中免疫器官中mRNA时空表达谱的有用工具。
