Nous remercions K. Gill de l&#39;aide pour l&#39;élevage de souris et de soins et de gestion de laboratoire. Merci également à Mukoyama membres du laboratoire de l&#39;aide technique. Le financement a été fourni par le Programme de recherche intra-muros des Instituts Naitonal de la Santé.

1. Collecte peau Limb embryonnaires de souris (E13.5 E17.5 ~) <ol><li> Euthanasier branchée femelles par une procédure approuvée. Selon notre protocole animales approuvées, les femelles sont euthanasiés par le CO 2 d&#39;exposition et ensuite assurée par dislocation cervicale. Lay l&#39;animal sur son papier absorbant et les tremper complètement dans 70% EtOH / H 2 O à partir d&#39;un flacon souple. </li><li> Disséquer l&#39;utérus intact et le placer dans un plat de 100 x 15 mm de Petri contenant une solution glacée Hanks Balanced Salt (HBSS) pour laver le sang. </li><li> Séparée et disséquer l&#39;embryon. Retirez l&#39;amnios très mince de l&#39;embryon. </li><li> (Option) Disséquer un seul embryon dans un plat de 35 x 10 mm de Petri, si chaque embryon doit être génotypés. La queue est disséqué transféré dans un tube de 0,2 ml par PCR pour le génotypage. </li><li> Coupez les pattes avant de l&#39;embryon et le transfert des membres antérieurs par une pince à anneau en 24 plaques à puits contenant 2 ml de glace froide paraformaldéhyde à 4% frais (PFA) dans un tampon phosphate salin (PBS). </li><li> Fixer les pattes avant avec mélange en douceur sur la table de mixage Nutator à 4 ° C pendant la nuit. </li><li> Le lendemain, retirer la PFA et laver trois fois pendant 5 min dans 2 ml de PBS avec mélange en douceur sur la table de mixage Nutator à température ambiante. </li><li> Transférer les pattes avant dans le méthanol à 100% (MeOH) et les stocker à -20 ° C l&#39;enzyme congélateur (le congélateur avec contrôle de température critique et sans fonction de dégivrage automatique). Les anticorps primaires énumérées dans le tableau 1 du travail après le traitement MeOH 100%. </li><li> Peler la peau hors de la patte avant en utilisant une pince à épiler fine. La peau du membre, quand déshydraté, devrait se séparent facilement de la branche. Placez d&#39;abord le membre avec la face ventrale (côté paume) vers le haut. Ensuite, en utilisant une pince à épiler fine, couper la peau comme le montre la vidéo. Suivant disséquer autour du membre entier, desquamation de la peau en douceur hors sans aucun dommage. </li></ol> 2. Whole-Mont coloration immunohistochimique de Skins Limb <ol><li> Réhydrater les peaux jambe en polypropylène à fond rond tube de 5ml en incubant à travers la série graduée de MeOH / PBT (PBS + 0,2% Triton X-100) (75%, 50%, 25%) pendant 5 min chacune. Notez que l&#39;échange de solutions doit être prudent pour éviter d&#39;endommager les peaux membre. </li><li> Laver deux fois pendant 5 min dans PBT avec mélange en douceur sur la table de mixage Nutator à température ambiante. </li><li> Bloquer les peaux de chèvre soit membre avec 10% de sérum / PBS 0,2% TX100 tampon de blocage des anticorps secondaire de chèvre ou de 10% de sérum d&#39;âne / PBS 0,2% TX100 tampon de blocage pour les anticorps secondaires âne pendant 2 heures en mélangeant doucement sur la table de mixage au Nutator la température ambiante. </li><li> Placez les peaux des membres sur un plat de 35 x 10 mm de Pétri et le transfert par une pince à anneau dans 2ml-microcentrifugeuse tube avec 800μl d&#39;anticorps primaire (dilution appropriée comme indiqué dans les tableaux) dans le tampon de blocage (soit 10% de sérum de chèvre / PBS 0,2 TX100% ou 10% de sérum d&#39;âne / PBS 0,2% TX100). Incuber les peaux membre avec mélange en douceur sur la table de mixage Nutator à 4 ° C pendant la nuit. Notez que plusieurs anticorps primaires provenant d&#39;espèces différentes (par exemple, anticorps monoclonal de rat + anticorps polyclonal de lapin) peuvent être utilisés simultanément. </li><li> Le lendemain, place les peaux des membres sur un plat de 35 x 10 mm et le transfert de Petri par une pince à anneau en polypropylène de 5 ml à fond rond tube avec 4 ml de tampon de lavage (soit 2% de sérum de chèvre / PBS 0,2% ou 2 TX100 % âne sérum / PBS 0,2% TX100). </li><li> Lavez cinq fois pendant 15 min en mélangeant doucement sur la table de mixage Nutator à température ambiante. </li><li> Placez les peaux des membres sur un plat de 35 x 10 mm de Pétri et le transfert par une pince à anneau dans 2ml-microcentrifugeuse tube avec 800μl d&#39;anticorps secondaire dans le tampon de blocage (soit 10% de sérum de chèvre / PBS 0,2% ou 10% TX100 âne sérum / PBS 0,2% TX100). Typiquement, nous utilisons des anticorps secondaires de Jackson ImmunoResearch (Cy3, Cy5, 1:300 dilutions) ou Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 dilution). Filtrer la solution d&#39;anticorps secondaire en utilisant les filtres 0.22μm PVDF seringue membrane pour éliminer les particules agrégées de l&#39;anticorps secondaire. Incuber les peaux des membres dans l&#39;obscurité ou enveloppés de papier d&#39;aluminium pendant 1 heure avec mélange en douceur sur la table de mixage Nutator à température ambiante. Notez que différents anticorps secondaires conjugués fluorescents provenant d&#39;espèces différentes peuvent être utilisées simultanément. </li><li> Placez les peaux des membres sur un plat de 35 x 10 mm de Pétri et le transfert par une pince à anneau en polypropylène à fond rond tube de 5ml avec 4ml de tampon de lavage (soit 2% de sérum de chèvre / PBS 0,2% ou 2% TX100 âne sérum / PBS 0,2% TX100). Lavez cinq fois pendant 15 min dans le noir ou enveloppés de papier d&#39;aluminium en mélangeant doucement sur la table de mixage Nutator à température ambiante. </li><li> (Option pour une contre-coloration contre le noyau) Incuber les peaux membre avec 4ml de tampon de lavage (soit 2% de sérum de chèvre / PBS 0,2% ou 2% TX100 âne sérum / PBS 0,2% TX100) avec A-Pro-3 (Invitrogen T3605 , 1:3000 dilution) dans le noir ou enveloppés de papier d&#39;aluminium pendant 10 min en mélangeant doucement sur la table de mixage à la salle Nutator temre. Ensuite, laver trois fois pendant 5 min avec 4ml de tampon de lavage (soit 2% de sérum de chèvre / PBS 0,2% ou 2% TX100 âne sérum / PBS 0,2% TX100) dans l&#39;obscurité ou enveloppés par des feuilles d&#39;aluminium en mélangeant doucement sur le le mélangeur Nutator à température ambiante. Notez que la coloration forte et spécifique pour les noyaux est observé pour TO-PRO-3 dans une émission spécifique (excitation HeNe nm 633). </li></ol> 3. Montage du Skins Limb à la diapositive <ol><li> Placez les peaux des membres sur un plat de 35 x 10 mm de Pétri. Retirez les poussières, cristaux, fibres de la couche interne de la peau en utilisant une pince à épiler amende en vertu du stéréomicroscope avec éclairage basse pour éviter la photo blanchissement. </li><li> Transférer les peaux branche à lame de microscope par un adhésif pinces. Placez les peaux avec la couche interne couché à la hausse sur la diapositive (c&#39;est à dire vers la lamelle). Aplatir les peaux attentivement l&#39;aide de pinces fines et retirer le report du tampon de lavage par Kimwipe. </li><li> Monter dans les médias anti-fade montage sans bulles d&#39;air. Nous utilisons 25 &quot;x25&quot; lamelle. Cure sur une surface plane dans l&#39;obscurité (par exemple, les échantillons montés à l&#39;aide de réactifs Prolongez l&#39;or sont placés la nuit dans l&#39;obscurité à température ambiante avant de visualisation). Pour stockage à long terme, sceller la lamelle sur la lame et stocker à 4 ° C. </li></ol> 4. Microscopie confocale <ol><li> Mettre en place des lasers appropriés pour les fluorophores. Nous utilisons Leica TCS SP5 microscope confocal avec trois sources laser Argon dont 488nm (pour Alexa Fluor 488 et GFP), DPSS 561nm (pour Alexa Fluor 568 et Cy3) et HeNe 633nm (pour Alexa Fluor 633, Cy5 et A-Pro-3) . </li><li> Utilisez l&#39;outil de scan séquentiel pour éviter ou réduire la diaphonie dans lequel tous les colorants dans les échantillons de double ou triple-tachés seront excités au même moment. Dans le mode de scan séquentiel, les images seront enregistrées dans un ordre séquentiel. </li><li> Des informations plus générales sur les colorants fluorescents et des lasers pour l&#39;excitation peut être créée en «microscopie confocale pour les biologistes» par Hibbs (2004) </li></ol> 5. Les résultats représentatifs Whole montage de microscopie confocale triple label immnofluorescence chez la souris forelimb peau à E15.5 avec des anticorps pour le marqueur de cellules endothéliales pan-PECAM-1 (figure 1A-C, D bleu), le marqueur de neurofilaments 2H3 (figure 1A vert), et le bon marqueur de cellules musculaires αSMA (figure 1A, B rouge) a révélé un aspect caractéristique de branchement des artères + αSMA, aligné avec 2H3 + nerfs périphériques dans la peau des membres. En plus de ramifications des vaisseaux sanguins, ce modèle vascularisation des membres peau est utilisée pour étudier la structuration des vaisseaux lymphatiques de branchement en utilisant des anticorps dirigés contre le marqueur de cellules endothéliales lymphatiques LYVE-1 (figure 1D, E rouge) et Neuropilin2 (NRP2) (figure 1D, F verte ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Figure 1" /> Figure 1. (Ca) Artères aligner avec les nerfs périphériques dans la peau membres de l&#39;embryon. Whole montage triple label microscopie confocale immnofluorescence avec des anticorps pour le marqueur de cellules endothéliales pan-PECAM-1 (AC, bleu), le marqueur de neurofilaments 2H3 (A, vert), et la douceur des cellules musculaires marqueurs αSMA (A et B, rouge ) est montré. À E15.5, 2H3 + nerfs périphériques (flèches ouvertes) associer à des artères (flèches) qui sont couverts par αSMA + cellules musculaires lisses. Vascularisation lymphatique (DF) dans la peau membres de l&#39;embryon. Triple-label microscopie confocale immnofluorescence avec des anticorps pour le marqueur de cellules endothéliales pan-PECAM-1 (D, bleu), le marqueur de cellules endothéliales lymphatiques LYVE-1 (D et E, rouge) et Neuropilin2 (NRP2) (D et F, vert ) est montré. Les vaisseaux lymphatiques sont visualisés par les deux LYVE-1 et NRP2, alors LYVE-1 est également exprimée par un sous-ensemble des macrophages (flèches ouvertes). Barre d&#39;échelle: 100 microns.

<ol>
	<li>Mukouyama, Y. S., Shin, D., Britsch, S., Taniguchi, M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+nerves+determine+the+pattern+of+arterial+differentiation+and+blood+vessel+branching+in+the+skin.">Sensory nerves determine the pattern of arterial differentiation and blood vessel branching in the skin.</a> Cell. 109, 693-705 (2002).</li><li>Mukouyama, Y. S., Gerber, H. P., Ferrara, N., Gu, C., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve-derived+VEGF+promotes+arterial+differentiation+via+neuropilin+1-mediated+positive+feedback.">Peripheral nerve-derived VEGF promotes arterial differentiation via neuropilin 1-mediated positive feedback.</a> Development. 132, 941-952 (2005).</li><li>Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+distinction+and+angiogenic+interaction+between+embryonic+arteries+and+veins+revealed+by+ephrin-B2+and+its+receptor+Eph-B4.">Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4.</a> Cell. 93, 741-753 (1998).</li><li>Gerety, S. S., Wang, H. U., Chen, Z. F., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetrical+mutant+phenotypes+of+the+receptor+EphB4+and+its+specific+transmembrane+ligand+ephrin-B2+in+cardiovascular+development.">Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.</a> Mol Cell. 4, 403-414 (1999).</li></ol>

Le système vasculaire est crucial pour le développement des organes pendant l&#39;embryogenèse ainsi que pour l&#39;entretien des organes et des fonctions de reproduction chez les adultes, car il fournit suffisamment d&#39;oxygène et de nutriments aux organes. Une bonne réseau vasculaire est bien établie avec des processus complexes et multi-étapes par l&#39;angiogenèse dans lequel pré-existante du réseau capillaire est réorganisée avec des structures très ramifiées et hiérarchique. Bien que de nombreux t...

Anticorps Marqueur des cellules endothéliales pan- <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> PECAM-1 </td><td> Hamster arménien (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> Dilution 1:100 # 1 </td></tr><tr><td> PECAM-1 </td><td> Rat (M) </td><td> BD </td><td> 553369 </td><td> 1:300 dilution </td></tr><tr><td> VEGFR2 </td><td> Rat (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> 1:200 dilution </td></tr><tr><td> CD34 </td><td> Rat (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 dilution </td></tr><tr><td> Le collagène IV </td><td> Lapin (P) </td><td> AbD Serotec </td><td> 2150-1470 </td><td> 1:300 dilution # 2 </td></tr></tbody></table> Artérielle marqueur des cellules endothéliales <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> Neuropilin1 </td><td> Lapin (P) </td><td> Le Kolodkin Alex Lab # 3 </td><td></td><td> 1:3000 dilution </td></tr><tr><td> Unc5H2 </td><td> Chèvre (P) </td><td> R &amp; D </td><td> AF1006 </td><td> 1:200 dilution </td></tr></tbody></table> Marqueur de cellules endothéliales veineuses <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> EphB4 </td><td> Chèvre (P) </td><td> R &amp; D </td><td> AF446 </td><td> Dilution 1:100 </td></tr></tbody></table> Marqueur des cellules endothéliales lymphatiques <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> LYVE-1 # 4 </td><td> Lapin (P) </td><td> Abcam </td><td> ab14917 </td><td> 1:200 dilution </td></tr><tr><td> LYVE-1 # 4 </td><td> Rat (M) </td><td> MBL </td><td> D225-3 </td><td> 1:200 dilution </td></tr><tr><td> Prox-1 </td><td> Lapin (P) </td><td> Chemicon </td><td> AB5475 </td><td> Dilution de 1:1000 </td></tr><tr><td> Prox-1 </td><td> Chèvre (P) </td><td> R &amp; D </td><td> AF2727 </td><td> Dilution 1:50 </td></tr><tr><td> Neuropilin2 </td><td> Lapin (P) </td><td> Signalisation cellulaire </td><td> 3366 </td><td> 1, 100 de dilution </td></tr><tr><td> Podoplanin </td><td> Hamster syrien (M) </td><td> Hybridomes Bank </td><td> 8.1.1 </td><td> 1:200 dilution </td></tr></tbody></table> Lisse des cellules musculaires / péricytes marqueur <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> αSMA-Cy3 </td><td> Souris (M) # 5 </td><td> Sigma </td><td> c-6198 </td><td> 1:500 dilution # 6 </td></tr><tr><td> NG2 </td><td> Lapin (P) </td><td> Chemicon </td><td> AB5320 </td><td> 1:200 dilution </td></tr><tr><td> SM22α </td><td> Lapin (P) </td><td> Abcam </td><td> ab14106 </td><td> 1:200 dilution </td></tr></tbody></table> Anticorps pour rapporteur GFP <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> GFP </td><td> Lapin (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 dilution </td></tr><tr><td> GFP </td><td> Rat (M) </td><td> Nacalai Tesque </td><td> 04404-84 </td><td> Dilution de 1:1000 </td></tr><tr><td> GFP </td><td> Chick (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 dilution </td></tr></tbody></table> Anticorps pour rapporteur LacZ <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> β-gal </td><td> Lapin (P) </td><td> MP Biomédical </td><td> 55976 </td><td> 1:5000 dilution </td></tr><tr><td> β-gal </td><td> Chèvre (P) </td><td> AbD Serotec </td><td> 4600-1409 </td><td> 1:500 dilution </td></tr><tr><td> β-gal </td><td> Chick (P) </td><td> Abcam </td><td> ab9361 </td><td> 1:200 dilution </td></tr></tbody></table> Anticorps pour axone périphériques <table border="1"><tbody><tr><td> Anticorps </td><td> Espèces </td><td> Société </td><td> Catalogue # </td><td> Conditions de travail </td></tr><tr><td> 2H3 </td><td> Souris (M) # 7 </td><td> Hybridomes Bank </td><td> 2H3 </td><td> 1:200 dilution </td></tr><tr><td> Tuj1 </td><td> Souris (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> 1:500 dilution </td></tr><tr><td> Périphérine </td><td> Lapin (P) </td><td> Chemicon </td><td> AB1530 </td><td> Dilution de 1:1000 </td></tr></tbody></table> Anticorps pour la migration des cellules de Schwann <table border="1"><tbody><tr><td> BFABP </td><td> Lapin (P) </td><td> Le laboratoire de Thomas Müller # 9 </td><td></td><td> 1:3000 dilution </td></tr></tbody></table> (P): anticorps polyclonal, (M): anticorps monoclonal # 1: chèvre anti-arménienne de hamster-Cy3 (Jackson ImmunoResearch 127-165-160) anticorps doit être utilisé comme anticorps secondaire. # 2: L&#39;anticorps de collagène IV peut être utilisé pour détecter des vaisseaux sanguins après l&#39;hybridation in situ. # 3: L&#39;anticorps Neuropilin1 est aimablement fourni par le laboratoire de l&#39;Kolodkin Alex à l&#39;Université Johns Hopkins. Sheep anticorps anti-humain Neuropilin1 est disponible en R &amp; D (AF3870), bien que nous n&#39;avons pas encore testé. # 4: Les anticorps LYVE-1 également détecter un sous-ensemble des macrophages dans la peau embryonnaire. # 5: L&#39;anticorps anti-IgG2a de souris αSMA est un anticorps monoclonal. # 6: L&#39;anticorps conjugué Cy3 αSMA est incubé pendant 1 heure à température ambiante avec s.Anticorps secondaires pour d&#39;autres anticorps primaires. # 7: 2H3 anticorps est la souris anticorps monoclonal IgG1 contre les neurofilaments. # 8: Tuj1 anticorps est IgG2a de souris d&#39;anticorps monoclonaux contre le neurone-spécifique de classe III bêta-tubuline. # 9: Le BFABP (cerveau-spécifiques d&#39;acides gras binding protein) antibody est aimablement fourni par le laboratoire de l&#39;Müller Thomas à Max-Delbrück-Center for Molecular Medicine.

whole-mount immunohistochemical analysis for embryonic limb skin vasculature: a model system to study vascular branching morphogenesis in embryo

Whole-Mont analyse immunohistochimique pour l&#39;imagerie du système vasculaire entier est essentielle pour la compréhension des mécanismes cellulaires de ramification morphogenèse. Nous avons développé le modèle de la vascularisation des membres de peau pour étudier le développement vasculaire dans lequel un pré-existante primitive plexus capillaire est réorganisée dans un réseau hiérarchiquement ramifiée vasculaire. Whole-Mont microscopie confocale avec étiquetage multiple permet pour l&#39;imagerie des vaisseaux sanguins robustes intactes ainsi que leurs composants cellulaires dont les cellules endothéliales, péricytes et cellules musculaires lisses, en utilisant des marqueurs fluorescents spécifiques. Les progrès dans ce modèle la vascularisation des membres peau avec des études génétiques ont permis d&#39;améliorer la compréhension des mécanismes moléculaires du développement vasculaire et les motifs. Le modèle vascularisation des membres peau a été utilisée pour étudier comment les nerfs périphériques fournissent un modèle spatial pour la différenciation et la structuration des artères. Cet article décrit un protocole vidéo simple et robuste pour colorer les vaisseaux sanguins intacts avec des anticorps spécifiques et vasculaires anticorps secondaires fluorescents, qui est applicable pour les organes embryonnaires vascularisées où nous sommes en mesure de suivre le processus du développement vasculaire.

Watch this Scientific Journal Video about Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo at JoVE.com

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Nous introduisons une immunohistochimie toute monture et laser microscopie confocale à balayage avec un étiquetage multiple pour analyser la formation de complexes réseau vasculaire dans la peau de souris membres de l&#39;embryon.

Whole-Mont analyse immunohistochimique à la vascularisation cutanée Limb embryonnaires: un modèle pour l&#39;étude vasculaire morphogenèse de branchement de l&#39;embryon

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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17.5K vues.  National Heart, Lung, and Blood Institute, National Institutes of Health. Nous introduisons une immunohistochimie toute monture et laser microscopie confocale à balayage avec un étiquetage multiple pour analyser la formation de complexes réseau vasculaire dans la peau de souris membres de l'embryon.

Vidéo: Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

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

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

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Biologie

Method for Whole Mount Antibody Staining in 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 ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Méthode de coloration Mont anticorps entier de Chick

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 ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself dispositif de récupération d&#39;échantillons cryoconservés tombé accidentellement dans les réservoirs de stockage cryogéniques

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Souris développement embryonnaire dans tout un système de culture d&#39;embryons sans sérum

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Analyse de l&#39;apoptose dans embryons de poissons zèbres par Whole montage immunofluorescence pour détecter caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...