Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae
Combined whole-mount fluorescence in situ hybridization and antibody staining in zebrafish embryos and larvae
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斑马鱼胚胎和幼鱼的整体荧光原位杂交和抗体染色相结合
DOI：
10.1038/s41596-020-0376-7
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发表时间：
2020-09-09
期刊：
影响因子：
14.8
通讯作者：
Luo, Lingfei
中科院分区：
文献类型：
--
作者：
He, Jianbo;Mo, Dashuang;Luo, Lingfei
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，同时检测多个mRNA和蛋白质在整个安装斑马鱼胚胎和幼虫。在我们的方法中，在IF之前进行FISH，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，使得该方案适用于整装胚胎和幼虫。通过使用PCR扩增的DNA模板和严格缓冲液减少脱靶杂交和FISH背景。该方案以高灵敏度同时检测多种mRNA和蛋白质，并能够以单细胞分辨率进行检测。该方案可以在6天内完成，克服了斑马鱼可用的可靠抗体的短缺，并利用斑马鱼研究器官发育和再生的优势。
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.