Manoli Martha, Driever Wolfgang
Developmental Biology, Faculty of Biology, University of Freiburg, Germany.
Cold Spring Harb Protoc. 2012 Aug 1;2012(8):pdb.prot069633. doi: 10.1101/pdb.prot069633.
This article describes a procedure for the dissociation of zebrafish (Danio rerio) embryos to produce a suspension of single cells that is suitable for fluorescence-activated cell sorting (FACS). The method has been applied to embryos at stages from 14 h post fertilization (hpf) to larvae at 5 d post fertilization (dpf), and it has also been successfully used for isolating fluorescently tagged neurons from whole dissociated embryos and early larvae. The cell collection procedures described in this protocol may also be adapted for older embryos and juvenile zebrafish. RNA can be extracted from the sorted cells and used for subsequent quantitative real-time PCR (qRT-PCR), microarrays, or next-generation sequencing (NGS) experiments.
本文介绍了一种解离斑马鱼（Danio rerio）胚胎以产生适用于荧光激活细胞分选（FACS）的单细胞悬液的方法。该方法已应用于受精后14小时（hpf）至受精后5天（dpf）的幼虫阶段的胚胎，并且也已成功用于从全部分离的胚胎和早期幼虫中分离荧光标记的神经元。本方案中描述的细胞收集程序也可适用于较老的胚胎和幼年斑马鱼。可以从分选的细胞中提取RNA，并用于后续的定量实时PCR（qRT-PCR）、微阵列或下一代测序（NGS）实验。
