A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.
A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.
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一组针对斑马鱼神经受体和分泌蛋白的重组单克隆抗体，适合整体免疫染色。
DOI：
10.1016/j.bbrc.2014.11.123
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发表时间：
2015
影响因子：
3.1
通讯作者：
Wright,GavinJ
中科院分区：
文献类型：
--
作者：
Staudt,Nicole;Müller-Sienerth,Nicole;Fane-Dremucheva,Alla;Yusaf,ShahnazP;Millrine,David;Wright,GavinJ
细胞表面受体和分泌蛋白在神经识别过程中发挥着重要作用，但由于它们的作用部位可能距离神经元细胞体很远，标记这些蛋白质的抗体对于了解它们的功能是有价值的。斑马鱼胚胎是神经生物学的一个流行的脊椎动物模型，但缺乏经过验证的抗体试剂。在这里，我们使用神经斑马鱼细胞表面的整个胞外区或在哺乳动物细胞中表达的分泌蛋白来选择针对十种不同抗原的单抗。用Western blotting对抗体进行鉴定，并测定其表位对福尔马林固定的敏感性。抗体的重排抗原结合区被扩增和克隆，从而能够从单个质粒中以重组形式表达。在福尔马林处理的斑马鱼胚胎大脑中，所有10种抗体都给出了特定的染色模式，表明这种普遍的方法特别有效地诱导抗体，在固定的整体组织中染色天然抗原。最后，我们表明，可以很容易地将额外的标签添加到重组抗体中，以便于多重染色。这里描述的抗体和方法将有助于解决斑马鱼研究中缺乏明确定义的抗体试剂的问题。
Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.
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DOI：
--
发表时间：
1988
期刊：
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DOI：
--
发表时间：
1987
期刊：
Biochemical and Biophysical Research Communications - BBRC
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影响因子：
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