Double in situ hybridization on mouse embryos for detection of overlapping regions of gene expression.
Double in situ hybridization on mouse embryos for detection of overlapping regions of gene expression.
复制标题
对小鼠胚胎进行双原位杂交，用于检测基因表达的重叠区域。
DOI：
10.1016/s0168-9525(96)90090-6
复制
发表时间：
1996
影响因子：
11.4
通讯作者：
J. Heath
中科院分区：
文献类型：
--
作者：
David Bueno;J. Skinner;H. Abud;J. Heath
在这种情况下，全载胚胎杂交是研究小鼠和其他脊椎动物发育调控基因表达的重要工具。提出了一种比较两个基因在同一胚胎中的表达模式和区分共表达区域的方法。使用2in探针，通常包括生物素或荧光素探针与地高辛探针联合使用。主要的困难是获得敏感和清晰可分辨的信号。使用针对地高辛、荧光素或生物素的荧光标记抗体，通常不能提供足够的灵敏度来检测反义RNA探针。使用传统的碱性磷酸酶或过氧化物酶底物来放大信号会出现2种染色颜色重叠的问题。描述了一种方案，允许使用全挂载杂交或全挂载杂交和切片染色的组合来可视化2个基因的表达和共表达。该方案类似于荧光素（FITC）和地高维新（DIG）标记的RNA探针杂交的描述程序，这些探针使用抗体-碱性磷酸酶偶联物进行检测，并将荧光染料与更常见的硝基蓝氯化四氮唑/5-溴-4-氯-3-吲哚磷酸甲苯胺盐（NBT/BCIP）染色相结合。将E11.5 d小鼠胚胎后肢芽与hoxd11探针（dig标记和酶标记荧光试剂盒染色）和hoxd13探针（fitc标记和NBT/BCIP染色）杂交。单光照交替显示两种染色颜色，而双光照同时显示两种染色颜色反应，从而可以适当地显示hoxd11和hoxd13共表达的区域。
In situhybridization of whole-mount embryos is an essential tool to study the expression of developmentally regulated genes in mouse and other vertebrates. A protocol is presented for comparison of the expression pattern of 2 genes in the same embryo, and distinguishing of co-expression areas. The use of 2in situprobes, usually involves either biotin or fluorescein probes in combination with digoxigenin probes. The major difficulty is to obtain sensitive and clearly distinguishable signals. The use of fluorescently-labelled antibodies against digoxigenin, fluorescein or biotin, does not usually provide enough sensitivity to detect antisense RNA probes. The use of conventional alkaline phosphatase or peroxidase substrates to amplify the signal presents problems where the 2 staining colours overlap. A scheme is described that allows visualization of the expression and co-expression of 2 genes using whole-mount hybridization or a combination of whole-mount hybridization and section staining. This protocol is similar to described procedures for hybridization of fluorescein (FITC)- and digoxigenin (DIG)-labelled RNA probes, which are detected using antibody-alkaline phosphatase conjugates, and combines a fluorochrome with the more usual nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt (NBT/BCIP) staining. A hindlimb bud of a E11.5 d mouse embryo was hybridized with aHoxd11probe (DIG-labelled and Enzyme Labelled Fluorescence kit stained) andHoxd13(FITC-labelled and NBT/BCIP stained). Single illuminations showed both staining colours alternately, whereas the double illumination showed both staining colour reactions simultaneously, allowing proper visualization of the areas whereHoxd11andHoxd13are co-expressed.