斑马鱼胚胎中亚细胞分辨率下单转录本的检测与自动分析

Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos.

作者信息

Stapel L Carine, Broaddus Coleman, Vastenhouw Nadine L

机构信息

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany.

Center for Systems Biology Dresden, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, 01307, Germany.

出版信息

Methods Mol Biol. 2018;1649:143-162. doi: 10.1007/978-1-4939-7213-5_9.

DOI:10.1007/978-1-4939-7213-5_9

PMID:29130195

Abstract

Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different cellular compartments resulting in quantitative information on transcript levels at subcellular resolution. The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish cryosections. The protocol includes a method to obtain high-quality sections of zebrafish embryos, an smFISH protocol optimized for zebrafish cryosections, and a user-friendly, automated analysis pipeline for cell segmentation and transcript detection. The software is freely available and can be used to analyze sections of any multicellular organism.

摘要

单分子荧光原位杂交（smFISH）是一种可视化单个mRNA分子的方法。当与细胞和细胞核分割相结合时，转录本可以被分配到不同的细胞区室，从而获得亚细胞分辨率下转录本水平的定量信息。由于缺乏针对多细胞生物体样本的实验方案和自动化图像分析流程，smFISH在斑马鱼中的应用受到了限制。在此，我们展示了一种针对斑马鱼冷冻切片进行smFISH的实验方案。该方案包括一种获取高质量斑马鱼胚胎切片的方法、一种针对斑马鱼冷冻切片优化的smFISH方案，以及一个用于细胞分割和转录本检测的用户友好型自动化分析流程。该软件可免费获取，可用于分析任何多细胞生物体的切片。