Name: application of the dna-specific stain methyl green in the fluorescent labeling of embryos
Uploaded: 2015-05-02T14:00:00.000+00:00
Duration: 5 min 59 s
Description: Watch this Scientific Journal Video about Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos at JoVE.com

The authors would like to acknowledge Patxi Jaso for the production of the video, PEDECIBA and Agencia Nacional de Investigaci&#243;n e Innovaci&#243;n (ANII) for partial funding.

1. A purificação do corante <ol><li> Prepara-se uma solução aquosa a 4% de verde de metilo por dissolução de 0,4 g de pó de mancha em 10 ml de água destilada. Misture bem até dissolver completamente. </li><li> Sob um fume-hood, misturá-lo com pelo menos 2 partes de clorofórmio. É importante verificar com antecedência se os tubos são resistentes ao clorofórmio. </li><li> Misturar bem e centrifuga-se durante 1 min a 2000 xg para acelerar a separação de fases. </li><li> Após centrifugação, a fase aquosa superior com verde de metilo e uma fase orgânica inferior com clorofórmio e o violeta de cristal de contaminantes são obtidos habitual. </li><li> Recuperar a fase superior e repita esta etapa até a fase mais baixa parece completamente desprovida de cristal violeta. </li><li> No final, a fase superior é recuperada e diluído em água até uma concentração final de 2% com verde de metilo. Esta solução de reserva é estável sobre a bancada durante meses e não necessita de ser protegido da luz. </li></ol><ol><li> Fixar os embriões durante a noite em 4% de formaldeído (paraformaldeído) a partir de em tampão fosfato salino (PBS). NOTA: Este pode levar desde algumas horas até durante a noite, dependendo da espessura dos embriões. No exemplo, nós fixa 48 horas pós-fertilização (HPF) embriões de peixe-zebra durante a noite. </li><li> Lavar os embriões três vezes em PBS-T (1% de Triton X-100 em PBS) durante 5 min. A concentração elevada de detergente permeabiliza a cutícula. </li><li> Prepara-se uma solução de 1: 10.000 de verde de metilo (a partir da solução stock 2%) em PBS-T: 5,000-1. A presença de detergente durante a incubação é útil para amostras de espessura, tais como embriões de peixe-zebra e larvas. </li><li> Incubar os embriões na solução, durante pelo menos 6 horas a 4 ° C, com agitação suave. Mais uma vez, a espessura do embrião é o parâmetro de calibração para o comprimento da incubação. </li><li> Lavar os embriões 3 times com PBS durante 30 min para remover o excesso de detergente. Metil fluorescência verde só é evidente quando ligado ao DNA, daí fundo da molécula não ligada é insignificante. </li><li> Montagem em um slide escavado ou de uma câmara caseira com solução de montagem (75% de glicerol, 0,1 M Tris-HCl pH 8). NOTA: Se a coloração nuclear deve ser realizada após imunomarcação, verde metilo pode ser incorporado à solução de montagem na concentração de trabalho, sem a necessidade de lavá-lo afastado. </li><li> Loja refrigerado ou iniciar imagiologia imediatamente. Realizar imagem com um confocal de varredura a laser (ou outra fluorescência) microscópio com excitação a 633 nm e emissão filtros definidos como registrar em 650-750 nm tendo em conta que metil emissão verde atinge o seu máximo em 677 nm. NOTA: Nós fornecemos os dados de medição do perfil de emissão verde de metilo, a ser carregados no software de aquisição, como material suplementar. </li></ol> 3. Fluorescent Nuclear Staining de Whole Pintainho embriões usando Metil Verde <ol><li> Incubar ovos de galinha fertilizados para a fase de interesse. Fixar os embriões durante a noite em 4% de formaldeído (paraformaldeído a partir de) em PBS a 4 ° C. Lavar os embriões 3 vezes em PBS-T, durante 2 h. </li><li> Prepara-se uma solução de 1: 10.000 de verde de metilo (a partir da solução stock 2%) em PBS-T: 5,000-1. Incubar os embriões na solução de coloração durante a noite a 4 ° C. Lavar os embriões 3 vezes em PBS, 1 h cada. Mount na câmara com 75% de glicerol, 0,1 M Tris, pH 8. </li></ol> 4. Criação de Imagens fluorescente etiquetado Núcleos em embriões inteiros montado <ol><li> Preparar uma câmara de imagem furando várias camadas de fita isolante sobre uma lâmina de microscópio e cuidadosamente cortar um buraco para ele com um bisturi para colocar os embriões dentro. Isso impedirá que os embriões de esmagamento durante o exame. </li><li> Transferir cuidadosamente os embriões para a câmara de imagiologia e encher a câmara com a parte superior com 75% de glicerol, Tris-HCl 0,1 M, pH = 8 para umaformação de bolhas vazio. </li><li> Cubra com um # 0 lamela evitar ou eliminar transbordante médio. Selá-lo com unha polonês. </li><li> Aquisição de imagens do espécime no microscópio de fluorescência equipado com um filtro de excitação de 633 ou linha de laser. NOTA: filtros de emissão deve cobrir o máximo de 677 emissões de verde metilo. </li></ol>

<ol>
	<li>Klonisch, T., Wark, L., Hombach-Klonisch, S., Mai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+imaging+in+three+dimensions:+a+unique+tool+in+cancer+research.">Nuclear imaging in three dimensions: a unique tool in cancer research.</a> Annals of anatomy. 192 (5), 292-301 (2010).</li><li>Kapuscinski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAPI:+a+DNA-specific+fluorescent+probe.">DAPI: a DNA-specific fluorescent probe.</a> Biotechnic. 70 (5), 220-233 (1995).</li><li>Latt, S. A., Stetten, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+studies+on+33258+Hoechst+and+related+bisbenzimidazole+dyes+useful+for+fluorescent+detection+of+deoxyribonucleic+acid+synthesis.">Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis.</a> The journal of histochemistry and cytochemistry. 24 (1), 24-33 (1976).</li><li>Van Hooijdonk, C. A., Glade, C. P., Van Erp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TO-PRO-3+iodide:+a+novel+HeNe+laser-excitable+DNA+stain+as+an+alternative+for+propidium+iodide+in+multiparameter+flow+cytometry.">TO-PRO-3 iodide: a novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry.</a> Cytometry. 17 (2), 185-189 (1994).</li><li>Beumer, T. L., Veenstra, G. J., Hage, W. J., Destr&#233;e, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemistry+on+Xenopus+embryos+using+far-red+fluorescent+dyes.">Whole-mount immunohistochemistry on Xenopus embryos using far-red fluorescent dyes.</a> Trends in genetics. 11 (1), 9 (1995).</li><li>Prieto, D., Aparicio, G., Morande, P. E., Zolessi, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast,+low+cost,+and+highly+efficient+fluorescent+DNA+labeling+method+using+methyl+green.">A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.</a> Histochemistry and cell biology. 142 (3), 335-345 (2014).</li><li>Kim, S. K., Nord&#233;n, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyl+green.+A+DNA+major-groove+binding+drug.">Methyl green. A DNA major-groove binding drug.</a> FEBS letters. 315 (1), 61-64 (1993).</li><li>Nord&#233;n, B., Tjerneld, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+methyl+green+to+deoxyribonucleic+acid+analyzed+by+linear+dichroism.">Binding of methyl green to deoxyribonucleic acid analyzed by linear dichroism.</a> Chemical Physics Letters. 50 (3), 508-512 (1977).</li><li>Lyon, H., Jakobsen, P., Andersen, a. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+methyl+green-pyronin+Y+procedures+using+pure+dyes.">Standardized methyl green-pyronin Y procedures using pure dyes.</a> The Histochemical journal. 18 (2-3), 90-94 (1986).</li><li>Amat, F., Keller, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+comprehensive+cell+lineage+reconstructions+in+complex+organisms+using+light-sheet+microscopy.">Towards comprehensive cell lineage reconstructions in complex organisms using light-sheet microscopy.</a> Development, growth &amp; differentiation. 55 (4), 563-578 (2013).</li><li>Chung, K., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLARITY+for+mapping+the+nervous+system.">CLARITY for mapping the nervous system.</a> Nature methods. 10 (6), 508-5013 (2013).</li><li>Lenz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+measurement+in+thick+tissue+layers+by+linear+or+nonlinear+long-wavelength+excitation.">Fluorescence measurement in thick tissue layers by linear or nonlinear long-wavelength excitation.</a> Applied. 38 (16), 3662-3669 (1999).</li></ol>

The authors have nothing to disclose.

The progressive development of microscopy techniques in the last decades, with a particular focus in variations of fluorescent microscopy, have led to the possibility of exploring the dynamics and structure of intact tissue samples, including whole embryos10,11. One major limitation has been, however, the availability of good quality fluorophores that can be used to directly and quickly stain subcellular structures. 
The most frequently used fluorescent DNA stains, such as DAPI or H...

Fluorescent staining of DNA is broadly and routinely used both in biological research and in clinical diagnosis, either for the detailed study of nuclear and chromosomal structure, for counterstaining tissues and cells, or for studying cell cycle parameters1. Although several DNA stains are available, the most widely used have been those excited by UV light and emitting in blue, which fit in the usual three-filter systems used in most conventional epifluorescence microscopes when combined with green and orange-red-emitting fluorophores, such as fluorescent proteins or small molecules attached to antibodies. Among these blue-emitting DNA stains, the most common are DAPI and Hoechst minor groove-binding agents2,3. Nonetheless, the incorporation of a wide variety of laser emission lines and spectral detection in laser scanning microscopes, has allowed researchers to choose for the use of far-red-emitting nuclear stains such as propidium iodide (PI) or TO-PRO 34. An advantage of these stains is that they overcome the problem of autofluorescence found in many cells and tissues, particularly in embryos5, but many of them have different problems such as having wide emission profiles or being very sensitive to photobleaching6.
The Swiss researcher Friedrich Miescher discovered DNA in 1869, extracting it from the nuclei of white blood cells found in pus. He called it nuclein, and showed a few years later that it formed precipitates with the relatively new stain methyl green. Methyl green is composed by three aniline rings, with different degrees of methylation, and has two positive charges that allow it to strongly bind to the major groove of DNA7,8. DNA staining by methyl green was very early shown to be specific, leading to the development by Unna and Pappenheim of a combined stain with pyronin, which specifically labels RNA. Methyl green-pyronin has since then been extensively used in routine histological technique9.
Until recently, very little was known about the fluorescent properties of methyl green. Our previous work, however, has uncovered some of methyl green spectral advantages6 such as its excitation at 633 nm, a convenient Stokes shift, its emission on the near-infrared portion of the electromagnetic spectrum, higher photostability than commercially available DNA stains with similar spectral properties and at a very low cost. This is why we decided to test it for being used as a very high affinity and specific fluorescent label for DNA on embryonic tissues and in whole embryos. Furthermore, until now methyl green nuclear staining was performed at low pH, making it difficult to combine with antibody staining. The goal of the method detailed here is to have a protocol for fluorescent nuclear DNA staining which overcomes the aforementioned inconvenience by performing the staining procedure at physiological pH, thus making it a suitable counterstain for immunolabeling on sections and whole tissues.

<table><tbody><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Methyl green</td><td>Dr. G. Gr&amp;uuml;bler</td><td></td><td>Also tested Sigma-Aldrich 323829, which is crystal violet-free</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Laser scanning confocal microscopy Leica TCS-SP5</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Phalloidin&amp;ndash;Tetramethylrhodamine B isothiocyanate</td><td>Sigma-Aldrich</td><td>&lt;br/&gt; P1951&amp;nbsp;</td><td></td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>372978</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>eppendorf</td><td>5811 000.010&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>microscope slides</td><td>Deltalab</td><td>D100004</td><td></td></tr><tr><td>cover slips</td><td>Esco optics</td><td>R525025</td><td></td></tr></tbody></table>

application of the dna-specific stain methyl green in the fluorescent labeling of embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A coloração nuclear de amostras grossas. O protocolo aqui descrito permite a realização de coloração homogênea de estruturas profundas no embriões inteiros. Lente desenvolvimento formar núcleos 48 hpf embriões de peixe-zebra são homogeneamente marcado (Figura 1). Coloração de ADN com verde de metilo permite a discriminação do ciclo celular dependentes diferenças morfológicas, tais como a identificação de figuras mitóticas ou núcleos apoptóticos ...

Watch this Scientific Journal Video about Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos at JoVE.com

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Aplicação do DNA-metil específico Stain Verde no Labeling fluorescentes de Embriões

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

application-dna-specific-stain-methyl-green-fluorescent-labeling

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

14.0K visualizações.  Institut Pasteur de Montevideo. A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Vídeo: Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Compreender Organogenesis precoce Usando um Simplificado<em&gt; In Situ</em&gt; Protocolo de hibridação em<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Biologia do Desenvolvimento

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Expressão de proteínas fluorescentes em<em&gt; Branchiostoma lanceolatum</em&gt; Por injecção de mRNA em oócitos não fertilizados

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Análise quantitativa da expressão da proteína para estudar Especificação Lineage no mouse Preimplantation Embriões

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

Multi-alvo Chromogenic Whole-mount<em&gt; In Situ</em&gt; Hibridização para Comparando Expressão Gênica em Domínios<em&gt; Drosophila</em&gt; Os embriões

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

Luz Folha com base em microscopia de fluorescência de Vida ou fixados e corados<em&gt; Tribolium castaneum</em&gt; Os embriões

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imagens ao vivo da motilidade celular e citoesqueleto de actina de neurônios individuais e células da crista neural em embriões Zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biologia

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Visualizando o tráfico dependente de citoesqueleto de organelas contendo lipídios em embriões Drosophila

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Usando Monte Whole In situ Hibridação a ligação de Biologia Molecular e Organísmicos

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

Rotulagem celular e injeção em Desenvolvimento embrionárias corações do rato

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Método para a rotulagem transcritos em células individuais Escherichia coli para único-molécula fluorescência In Situ as experiências de hibridação

Aplicação do DNA-metil específico Stain Verde no Labeling fluorescentes de Embriões

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