Abstract
A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Large-scale developmental screens such as unbiased mutagenesis surveys have been limited in their applicability to embryonic systems, as the technologies for quantifying precise expression patterns in whole animals has not kept pace with other sequencing-based technologies. Here, we outline an open-source semi-automated pipeline to chemically fixate, stain, and 3D-image Drosophila embryos. Central to this pipeline is a liquid handling robot, Flyspresso, which automates the steps of classical embryo fixation and staining. We provide the schematics and an overview of the technology for an engineer or someone equivalently trained to reproduce and further improve upon Flyspresso, and highlight the Drosophila embryo fixation and colorimetric or antibody staining protocols. Additionally, we provide a detailed overview and stepwise protocol for our adaptive-feedback pipeline for automated embryo imaging on confocal microscopes. We demonstrate the efficiency of this pipeline compared to classical techniques, and how it can be repurposed or scaled to other protocols and biological systems. We hope our pipeline will serve as a platform for future research, allowing a broader community of users to build, execute, and share similar experiments.
Highlights
A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts
We developed Flyspresso, a customizable syringe-based microplate washer to carry out Drosophila embryo experiments at a higher throughput, and an adaptive feedback confocal microscope pipeline that allows the automated acquisition of samples[10]
Our pipeline automates several critical steps: (1) embryo fixation, (2) vitelline membrane removal, (3) antibody or chemical staining of embryos, and (4) the automated imaging of embryos. The goal of this approach was to streamline classical Drosophila embryo fixation, immunohistochemistry, and imaging protocols on 24 embryonic samples per experiment—embracing a “Do It Yourself ” (DIY) ethos that could be adapted by others[9, 11]
Summary
A significant challenge for developmental systems biology is balancing throughput with controlled conditions that minimize experimental artifacts. Automated liquid-handling robots can precisely and accurately screen multiple whole-mount samples in parallel[8, 9] These systems exist for embryo staining, they have several shortcomings, including manual embryo fixations, are prohibitively expensive for many groups, and are not scalable or adaptable. Our pipeline automates several critical steps: (1) embryo fixation, (2) vitelline membrane removal, (3) antibody or chemical staining of embryos, and (4) the automated imaging of embryos The goal of this approach was to streamline classical Drosophila embryo fixation, immunohistochemistry, and imaging protocols on 24 embryonic samples per experiment—embracing a “Do It Yourself ” (DIY) ethos that could be adapted by others[9, 11]
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