Les anticorps monoclonaux (15.3B9, PAX7) développé par (J. Dodd, MC Jessell et A. Kawakami) ont été obtenus auprès de la Banque de développement d&#39;hybridomes études élaborées sous les auspices de l&#39;NICHD et maintenu par l&#39;Université de l&#39;Iowa, Département des sciences biologiques , dans l&#39;Iowa. DP est récipiendaire du Prix Ruth Kirschstein 1F32-01A1 DA021977 du National Institute on Drug Abuse. Ce travail a été soutenu par la Fondation Margaret M. Alkek au RHF.

I. Schéma Aperçu: Cette vidéo montre les différentes étapes de l&#39;immunohistochimie monter ensemble dans l&#39;embryon de poulet. Tout d&#39;abord, l&#39;embryon est fixé en PFA [IHC1]. Ensuite, l&#39;activité peroxydase endogène est éteint [IHC2]. L&#39;embryon est ensuite incubée dans l&#39;anticorps primaire [IHC3]. Après plusieurs lavages, l&#39;embryon est incubé dans anticorps secondaire [IHC4]; réaction colorée se révèle en DAB [IHC5] et la coloration des anticorps apparaît orange [IHC6]. <img src="/files/ftp_upload/old/956_2009_1_26_3/figure 1.png" alt="Figure 1" width="600" height="573" /> Partie 1: Fixation des embryons <ol><li> Pour effectuer immunohistochimie monter ensemble sur les embryons de poulet, d&#39;abord ouvrir un oeuf en tapant la coquille avec une pince et enlever des morceaux de la coquille. </li><li> Retirez l&#39;albumine épaisse avec une pince, et de l&#39;inclinaison du sac vitellin avec des pinces grossières pour que l&#39;embryon tournée vers le haut. </li><li> Avec des ciseaux fins, découpez un carré de vitellus autour de l&#39;embryon. Retirez l&#39;embryon du jaune avec une cuillère, et les placer dans un plat contenant du PBS. </li><li> Sous un microscope de dissection, retirer soigneusement les membranes et le jaune et le transfert d&#39;embryon dans un plat frais contenant du PBS. </li><li> Pin de l&#39;embryon vers le bas avec des pinces et des épingles d&#39;insectes, et aspirer le PBS. Remplacez cela avec 4% en PBS PFA, et permettent de fixer l&#39;embryon 1h à température ambiante. </li></ol> Partie 2: Préparation des embryons pour l&#39;étape d&#39;anticorps <ol><li> Pour minimiser la contamination microbienne potentielle, utilisez ddH 2 O dans toutes les étapes suivantes. </li><li> Après l&#39;embryon est fixe, aspirer la solution fixateur et éliminer correctement les déchets chimiques. Remplir le plat avec du PBS. </li><li> Ensuite, en utilisant un couteau de microdissection, couper un carré autour de l&#39;embryon pour enlever les membranes extra-embryonnaires. Retirer les épingles insectes avec une pince. </li><li> Après les broches ont été enlevées, utiliser une pipette Pasteur émoussée fin de transférer l&#39;embryon d&#39;un flacon à scintillation contenant des PBT (PBS pH 7,4, 0,5% Triton X). </li><li> Laver l&#39;embryon avec PBT, 3 fois pendant 10 minutes chacun. </li><li> Retirer PBT du flacon à scintillation, et la remplacer par PbTx contenant 0,3% de H 2 O 2 afin d&#39;inactiver peroxydase endogène potentiel. </li><li> Incuber pendant 2 heures à température ambiante sur les nutator. </li><li> Lavez embryon avec PBT, 3 fois pendant 10 minutes chacun, puis 3 fois pendant 30 minutes chacun. </li></ol> Partie 3: l&#39;incubation d&#39;anticorps <ol><li> Tacher l&#39;embryon avec l&#39;anticorps, commencez par laver l&#39;embryon pendant une heure dans un tampon de blocage ou de 1% de BSA / END 1% / PBT (nous utilisons NGS parce que les anticorps secondaire est soulevée dans de chèvre). Incuber sur nutator pendant une heure à température ambiante. </li><li> Diluer l&#39;anticorps primaire 1:01 dans un tampon de blocage, et l&#39;embryon incuber dans cette solution pendant 2 jours à 4 ° C sur nutator. Primaire facteur de dilution d&#39;anticorps dépend du choix de l&#39;anticorps primaire. Ici, nous utilisons un anticorps de la Banque de développement d&#39;études d&#39;hybridome, qui est fourni surnageant. Nous diluer 1:1 dans un tampon bloquant. </li><li> Ensuite, effectuez trois de 10 minutes lavages en PBT, suivis de 3 en 1 heure lavages en PBT. </li><li> Après le lavage, diluer l&#39;anticorps secondaire 1:2500 dans le tampon de blocage (dans ce cas, conjuguée à la peroxydase-chèvre anti-souris IgG (H + L), et de l&#39;embryon incuber dans cette solution S / N à 4 ° C sur nutator. </li><li> Effectuer 3 10 minutes lavages en PBT, suivis de 3 en 1 heure lavages en PBT. </li></ol> Partie 3: réaction colorée <ol><li> Pour la réaction de couleur de l&#39;embryon taché, d&#39;abord effectuer deux lavages de 20 minutes dans du tampon Tris (Tris HCl 100 mM, pH 7,4). </li><li> En attendant la dissolution du substrat DAB (3,3 &#39;-diaminobenzidine tétrachlorhydrate) dans un tampon Tris à 500μg/ml sous une hotte; garder solution dans l&#39;obscurité, sur la glace. </li><li> Enlever le dernier tampon de lavage Tris partir de l&#39;embryon flacon contenant et remplacer avec une solution DAB 5ml de l&#39;étape 3.2. Gardez à l&#39;sombres sur nutator pendant 20 mn. Eliminer Eppendorf pointe dans un seau contenant une solution javellisée à 10% pour décontaminer DAB. </li><li> Pendant ce temps préparer un stock de 0,3% H 2 O 2 en DH 2 O sur la glace. </li><li> Ajouter le bouillon de 50 pl H 2 O 2 à fiole contenant des embryons en DAB. Gardez à l&#39;obscurité. Après 1-2 minutes, surveiller la réaction sous le microscope. </li></ol> Partie 4: le traitement des embryons pour la photographie et de l&#39;histologie <ol><li> Lorsque la réaction colorée est complet, jetez-DAB dans un seau l&#39;eau de Javel. Remplacez-le par 5 ml d&#39;eau du robinet. </li><li> Effectuer deux 10 minutes lavages en PBS. </li><li> Pour traiter de la photographie et le sectionnement de la cire, déshydrater dans une série d&#39;éthanol (25%, 50%, 75% et 100%) pendant 10 minutes chacun. </li><li> Remplacer l&#39;éthanol avec 0,5 - 1 ml d&#39;huile de bois de cèdre (cela va rendre le translucides embryon). Processus pour la photographie dans l&#39;huile de bois de cèdre. </li><li> Après la photographie, le retour d&#39;embryons pour flacon et remplacer l&#39;huile de bois de cèdre avec de l&#39;éthanol à 100%. Répétez l&#39;étape d&#39;éthanol. </li><li> Remplacer ethanol avec 5% vert solide FCF rapide dans de l&#39;éthanol à 100%, et incuber pendant 3 minutes (cette étape fera embryons visibles pour l&#39;histologie). Remplacer Rapide solution verte FCF avec de l&#39;éthanol 100% et un processus pour l&#39;histologie. </li></ol> Les résultats représentatifs: <img src="/files/ftp_upload/old/956_2009_1_26_2/figure2.png" alt="Figure 2" width="600" height="498" /> Dans les exemples ci-dessous, les embryons sont disséqués à des stades HH 10 (A), 12 (B) et 11 (C), les embryons montrent une expression de la PAX 7 dans les pays émergents Crestas neurones ainsi que dans les somites et le tube neural (A, B); En (C), la notochorde est étiqueté avec 15.3B9 (Pas-1) (anticorps fournis par la Banque de développement d&#39;études d&#39;hybridome).

<ol>
	<li>Yamada, T., Placzek, M., Tanaka, H., Dodd, J., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+developing+nervous+system:+Polarizing+activity+of+the+floor+plate+and+notochord.">Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord.</a> Cell. 64, 635-647 (1991).</li><li>Streit, A., Stern, C. D., Thery, C., Ireland, G. W., Aparicio, S., Sharpe, M. J., Gherardi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+HGF/SF+in+neural+induction+and+its+expression+in+Hensen's+node+during+gastrulation.">A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation.</a> Development. 121, 813-824 (1995).</li><li>Basch, M., Bronner-Fraser, M., Garcia-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li></ol>

Cette vidéo montre les différentes étapes dans l&#39;exécution de coloration des anticorps entiers monter dans des embryons de poulet jeunes. Ce protocole est essentiellement utilisé pour la caractérisation spatiale et temporelle de nouveaux anticorps chez le poulet 2,3, ainsi que pour l&#39;utilisation de marqueurs antigéniques connues pour déterminer les malformations embryonnaires suivantes insulte 4.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH4 (16 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Other</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td> Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to cut embryo from surrounding membranes following fixation</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>30% H2O2</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1009</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3803</td>
<td>&nbsp;</td>
<tr>
<td>NGS</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>005-000-121</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibodies</td>
<td>Reagent</td>
<td>Developmental studies Hybridoma Bank</td>
<td>4G11, 15.3B9, PAX7</td>
<td>For these antibodies, investgators used mouse donnors</td>
</tr>
<tr>
<td>Peroxidase conjugated-Goat Anti-Mouse IgG (H+L) </td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>115-035-003</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride</td>
<td>Reagent</td>
<td>Pierce</td>
<td>34001</td>
<td>Store at -20; Allow bottle to warm to RT before use.</td>
</tr>
<tr>
<td>Cedar wood oil</td>
<td>Reagent</td>
<td>Sigma</td>
<td>W522503</td>
<td>&nbsp;</td>
<tr>
<td>Fast green FCF</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

method for whole mount antibody staining in chick

L&#39;embryon de poulet est un outil précieux dans l&#39;étude du développement embryonnaire précoce. Sa transparence, l&#39;accessibilité et la facilité de manipulation, en font un outil idéal pour étudier l&#39;expression d&#39;anticorps dans le développement du cerveau, du tube neural et des somites. Cette vidéo montre les différentes étapes de coloration des anticorps entiers de montage en utilisant HRP conjugué anticorps secondaire; d&#39;abord, l&#39;embryon est disséquée de l&#39;oeuf et fixés dans du paraformaldéhyde. Deuxièmement, la peroxydase endogène est inactivé; L&#39;embryon est ensuite exposée à l&#39;anticorps primaire. Après plusieurs lavages, l&#39;embryon est incubée avec un anticorps secondaire conjugué à la HRP. L&#39;activité peroxydase est révélée en utilisant la réaction avec le substrat diaminobenzidine. Enfin, l&#39;embryon est fixe et traitées pour la photographie et de sectionnement. L&#39;avantage de cette méthode sur l&#39;utilisation d&#39;anticorps fluorescents est que les embryons peuvent être traités pour la coupe de la cire, permettant ainsi à l&#39;étude de sites antigéniques en coupe transversale. Cette méthode a été initialement présenté par Jane et Tom Dodd Jessell<sup&gt; 1</sup&gt;.

Watch this Scientific Journal Video about Method for Whole Mount Antibody Staining in Chick at JoVE.com

Method for Whole Mount Antibody Staining in Chick

Cette vidéo montre tout le mont immunohistochimie, une méthode par laquelle le profil d&#39;expression spatiale et temporelle d&#39;un antigène peut être visualisé dans des embryons de poulet jeunes. Cette méthode a été initialement présenté par Jane et Tom Dodd Jessell.

Méthode de coloration Mont anticorps entier de Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

method-for-whole-mount-antibody-staining-in-chick

Research

JoVE Journal

Biology

15.7K vues.  Texas A&M University (TAMU). Cette vidéo montre tout le mont immunohistochimie, une méthode par laquelle le profil d'expression spatiale et temporelle d'un antigène peut être visualisé dans des embryons de poulet jeunes. Cette méthode a été initialement présenté par Jane et Tom Dodd Jessell.

Vidéo: Method for Whole Mount Antibody Staining in Chick

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

Recherche

Biologie

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish entier Mont haute résolution double fluorescent In Situ Hybridation

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mont In Situ</emHybridation> de E8.5 à E11.5 embryons de souris

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Toute Mont ARN fluorescent<em&gt; In situ</emHybridation&gt; d&#39;<em&gt; Drosophila</em&gt; Embryons

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

ARN<em&gt; In situ</emHybridation&gt; en embryons entiers de montage et histologie Adapté pour les élasmobranches marins

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Anticorps coloration dans<em&gt; C. Elegans</em&gt; Utilisation de «gel-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Une méthode efficace pour quantitative, analyse unicellulaire de la chromatine Modification et architecture nucléaire dans l&#39;ensemble du montage ovules dans<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Appartement Mont Préparation pour l&#39;observation et l&#39;analyse des échantillons embryon de poisson zèbre souillé par toute la montagne<em&gt; In situ</em&gt; Hybridation

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

A Mont entier<em&gt; In Situ</em&gt; Méthode Hybridation pour le mollusque gastéropode<em&gt; Lymnaea stagnalis</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

Protocole CUBIC Visualise Protein Expression à cellule unique Résolution dans les préparations entières de la peau de montage

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...