Abstract
ABSTRACTZebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.
Highlights
The increase in zebrafish genomic resources together with more sophisticated protocols for genome editing and other tools have contributed to unravel the genetic networks controlling development, and generated zebrafish models with relevance to human disease
For a detailed phenotypic characterization, especially for internal structures, and/ or structures that develop beyond the stage of complete transparency of the embryo, such as the skeletal system, it is helpful to complement whole-mount techniques and standard embedding and sectioning procedures, including paraffin or vibratome sections
We demonstrate that glycol methacrylate (GMA) embedding can be part of daily routine, yielding cellular details with high resolution, nicely complementing methods that rely on observations of whole-mount embryos and providing a substitute if advanced imaging technology is not readily available
Summary
The increase in zebrafish genomic resources together with more sophisticated protocols for genome editing and other tools have contributed to unravel the genetic networks controlling development, and generated zebrafish models with relevance to human disease (e.g. skeletal diseases, Laizé et al, 2014; Witten et al, 2017; Gistelinck et al, 2018). For a detailed phenotypic characterization, especially for internal structures, and/ or structures that develop beyond the stage of complete transparency of the embryo, such as the skeletal system, it is helpful to complement whole-mount techniques and standard embedding and sectioning procedures, including paraffin or vibratome sections Advanced imaging techniques such as dual photon microscopy or light sheet microscopy can overcome the limitations of observations on whole-mount embryos or superficially positioned structures. They have the advantage of analysis in 3D, and enable in vivo analysis which can readily and precisely answer a broad range of biological questions, including those regarding dynamic cell movement While standard histological techniques provide a convenient way to analyze tissues and cells, they need to be adapted for the use of fluorophores in zebrafish research
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