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Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae.
Nature Protocols ( IF 14.8 ) Pub Date : 2020-09-09 , DOI: 10.1038/s41596-020-0376-7
Jianbo He 1 , Dashuang Mo 1 , Jingying Chen 1 , Lingfei Luo 1
Affiliation  

RNA fluorescence in situ hybridization (FISH) and antibody staining/immunofluorescence (IF) are widely used to detect distributions of mRNAs and proteins. Here we describe a combined FISH and IF protocol to simultaneously detect multiple mRNAs and proteins in whole-mount zebrafish embryos and larvae. In our approach, FISH is performed before IF to prevent mRNA degradation during the IF procedure. Instead of proteinase K digestion, Triton X-100 treatment and skin removal are used to permeate tissues and preserve antigen epitopes, making this protocol applicable to both whole-mount embryos and larvae. Off-target hybridization and FISH background are reduced by using PCR-amplified DNA templates and stringent buffers. This protocol simultaneously detects multiple mRNAs and proteins with high sensitivity, and enables detection at single-cell resolution. The protocol can be completed within 6 days, overcoming the shortage of reliable antibodies available for zebrafish and exploiting the advantages of zebrafish for studying organ development and regeneration.



中文翻译:

在斑马鱼胚胎和幼虫中结合全贴装荧光原位杂交和抗体染色。

RNA 荧光原位杂交 (FISH) 和抗体染色/免疫荧光 (IF) 被广泛用于检测 mRNA 和蛋白质的分布。在这里,我们描述了一个组合的 FISH 和 IF 协议,以同时检测整个斑马鱼胚胎和幼虫中的多个 mRNA 和蛋白质。在我们的方法中,FISH 在 IF 之前进行,以防止 IF 过程中的 mRNA 降解。代替蛋白酶 K 消化,Triton X-100 处理和皮肤去除用于渗透组织并保留抗原表位,使该协议适用于整体胚胎和幼虫。通过使用 PCR 扩增的 DNA 模板和严格的缓冲液,可以减少脱靶杂交和 FISH 背景。该协议以高灵敏度同时检测多个 mRNA 和蛋白质,并能够以单细胞分辨率进行检测。

更新日期:2020-09-10
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