Wir danken K. Gill für die Unterstützung bei der Maus Zucht und Pflege sowie für Labor-Management. Danke auch an Mukoyama lab Mitglieder für technische Hilfe. Die Finanzierung wurde von Intramural Research Program der Nationalfeiertag Institutes of Health zur Verfügung gestellt.

1. Sammeln embryonalen Maus-Limb Skin (E13.5 ~ E17.5) <ol><li> Euthanize gesteckt Weibchen durch zugelassene Verfahren. Nach unseren zugelassenen Tier-Protokoll sind die Weibchen von CO 2 Belastung eingeschläfert und dann durch Genickbruch gewährleistet. Legen Sie das Tier auf seinen saugfähigen Papiertuch und legen Sie ihn gründlich in 70% EtOH / H 2 O aus einer Spritzflasche. </li><li> Präparieren Sie die Gebärmutter intakt und legen Sie sie in einem 100 x 15 mm Petrischale mit eiskaltem Hanks Balanced Salt Solution (HBSS) zu waschen Blut. </li><li> Separate und sezieren des Embryos. Entfernen Sie die sehr dünne Amnion aus dem Embryo. </li><li> (Option) Dissect eines einzigen Embryos in einem 35 x 10 mm Petrischale, wenn jeder Embryo muss genotypisiert werden. Dissected Schwanz ist ein 0,2 ml PCR-Röhrchen für die Genotypisierung übertragen. </li><li> Schneiden Sie die Vorderbeine des Embryos und Transfer Vorderbeine durch einen Ring Pinzette in 24-Well-Platte mit 2 ml eiskaltem frischen 4% Paraformaldehyd (PFA) in Phosphatpuffer Saline (PBS). </li><li> Fix die Vorderbeine mit sanftem Mischen auf dem Kolbenpendel Mixer bei 4 ° C über Nacht. </li><li> Am folgenden Tag, entfernen Sie die PFA und waschen dreimal für 5 min in 2 ml PBS mit sanftem Mischen auf dem Kolbenpendel Mixer bei Raumtemperatur. </li><li> Übertragen Sie die Vorderbeine in 100% Methanol (MeOH) und speichert sie bei -20 ° C Enzym Gefrierschrank (Gefrierschrank mit kritischen Temperatur-Steuerung und ohne automatische Abtauung). Primärantikörper in Tabelle 1 der Arbeit nach der 100% MeOH Behandlung aufgeführt. </li><li> Peel off Haut aus dem Vorderbein mit feinen Pinzette. Limb Haut, wenn entwässert, sollte einfach zu trennen von der Extremität. Der erste Platz der Extremität mit Bauchseite (Handfläche) nach oben. Dann mit feinen Pinzetten, schneiden Sie die Haut wie im Video gezeigt. Next um den gesamten Leib sezieren, Peeling die Haut sanft, ohne Schäden. </li></ol> 2. Whole-mount immunhistochemische Färbung von Limb Skins <ol><li> Rehydrieren die Extremität Skins in 5ml Polypropylen mit rundem Boden Rohr durch Inkubation durch die abgestufte Reihe von MeOH / PBT (PBS + 0,2% Triton X-100) (75%, 50%, 25%) für jeweils 5 min. Beachten Sie, dass der Austausch-Lösungen sollten vorsichtig sein, um eine Beschädigung des Körpers skins. </li><li> Zweimal waschen für 5 min in PBT mit sanftem Mischen auf dem Kolbenpendel Mixer bei Raumtemperatur. </li><li> Blockieren Sie die Gliedmaßen Skins entweder mit 10% Ziegenserum / PBS +0,2% TX100 Blockierungspuffer für Ziege Sekundärantikörper oder 10% Donkey Serum / PBS +0,2% TX100 Blockierungspuffer für Esel Sekundärantikörper für 2 Stunden unter leichtem Mischen auf dem Kolbenpendel Mixer bei Raumtemperatur. </li><li> Legen Sie das Bein Skins auf einem 35 x 10 mm Petrischale und Transfer von einem Ring Pinzette in 2ml-Reaktionsgefäß mit 800μl des primären Antikörpern (entsprechender Verdünnung in den Tabellen aufgeführt) in den Blocking-Puffer (entweder 10% Ziegenserum / PBS + 0,2 % TX100 oder 10% Donkey Serum / PBS +0,2% TX100). Inkubieren der Extremität Skins mit sanftem Mischen auf dem Kolbenpendel Mixer bei 4 ° C über Nacht. Beachten Sie, dass mehrere primäre Antikörper aus verschiedenen Spezies (zB Ratte monoklonale Antikörper + polyklonalen Kaninchen-Antikörper) abgeleitet gleichzeitig genutzt werden können. </li><li> Am folgenden Tag, Ort der Extremität Skins auf einem 35 x 10 mm Petrischale und Transfer von einem Ring Pinzette in 5ml Polypropylen mit rundem Boden Rohr mit 4 ml Waschpuffer (entweder 2% Ziegenserum / PBS +0,2% TX100 oder 2 % Donkey Serum / PBS +0,2% TX100). </li><li> Wash fünfmal für 15 min unter leichtem Mischen auf dem Kolbenpendel Mixer bei Raumtemperatur. </li><li> Legen Sie das Bein Skins auf einem 35 x 10 mm Petrischale und Transfer von einem Ring Pinzette in 2ml-Reaktionsgefäß mit 800μl der Sekundärantikörper in den Blocking-Puffer (entweder 10% Ziegenserum / PBS +0,2% TX100 oder 10% Donkey Serum / PBS +0,2% TX100). In der Regel verwenden wir Sekundärantikörper von Jackson ImmunoResearch (Cy3, Cy5, 1:300 Verdünnungen) oder Invitrogen (Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 633, 1:250 Verdünnung). Filtern Sie die sekundären Antikörper-Lösung mit 0,22 &amp;mgr; m PVDF-Membran Spritzenfilter, um aggregierte Partikel der sekundären Antikörper zu entfernen. Inkubieren der Extremität Skins im Dunkeln oder mit Alufolie umwickelt für 1 Stunde mit leichtem Rühren auf dem Kolbenpendel Mixer bei Raumtemperatur. Beachten Sie, dass verschiedene fluoreszierende konjugierte sekundäre Antikörper von verschiedenen Spezies abgeleitet gleichzeitig genutzt werden können. </li><li> Legen Sie das Bein Skins auf einem 35 x 10 mm Petrischale und Transfer von einem Ring Pinzette in 5ml Polypropylen mit rundem Boden Rohr mit 4 ml Waschpuffer (entweder 2% Ziegenserum / PBS +0,2% TX100 oder 2% Donkey Serum / PBS +0,2% TX100). Wash fünfmal für 15 min im Dunkeln oder umwickelt mit Alufolie mit leichtem Rühren auf dem Kolbenpendel Mixer bei Raumtemperatur. </li><li> (Option für die Gegenfärbung gegen Kern) Inkubieren der Extremität Skins mit 4 ml Waschpuffer (entweder 2% Ziegenserum / PBS +0,2% TX100 oder 2% Donkey Serum / PBS +0,2% TX100) mit To-Pro-3 (Invitrogen T3605 , 1:3000 Verdünnung) in der Dunkelheit oder umwickelt mit Alufolie für 10 min unter leichtem Mischen auf dem Kolbenpendel Mixer bei Zimmertemperatur Temre. Dann waschen dreimal 5 min mit 4 ml Waschpuffer (entweder 2% Ziegenserum / PBS +0,2% TX100 oder 2% Donkey Serum / PBS +0,2% TX100) im Dunkeln oder durch Aluminium-Folie mit leichtem Rühren auf gewickelt dem Kolbenpendel Mixer bei Raumtemperatur. Beachten Sie, dass starke und spezifische Färbung für Keime für Um-pro-3 ist in einer spezifischen Emission (HeNe 633 nm Anregung) beobachtet. </li></ol> 3. Montage des Limb Skins auf Slide <ol><li> Legen Sie das Bein Skins auf einem 35 x 10 mm Petrischale. Entfernen Sie Staub, Kristallen, Fasern von der inneren Schicht der Haut mit feinen Pinzetten unter dem Stereomikroskop mit geringer Beleuchtung auf umfangreiche Foto Bleichen zu vermeiden. </li><li> Übertragen Sie die Extremitäten Skins, um Klebstoff Objektträger durch einen Ring Pinzette. Legen Sie die Haut mit der inneren Schicht liegt oben auf der Folie (dh in Richtung Deckglas). Flatten die Felle sorgfältig mit feinen Pinzette und entfernen carry-over Waschpuffer durch Kimwipe. </li><li> Berg in anti-fade Montage Medien ohne Luftblasen. Wir verwenden 25 &quot;x25&quot; Deckglas. Cure auf einer ebenen Fläche in der Dunkelheit (zB montiert die Proben unter Verwendung ProLong Gold-Reagenz werden über Nacht im Dunkeln bei Raumtemperatur vorgelegt, bevor viewing). Für die langfristige Lagerung, Deckglasränder, um die Folie und lagern bei 4 ° C. </li></ol> 4. Konfokale Mikroskopie <ol><li> Richten Sie entsprechende Laser für Fluorophore. Wir verwenden Leica TCS SP5 konfokalen Mikroskop mit drei Laserquellen mit Argon 488nm (für Alexa Fluor 488 und GFP), DPSS 561nm (für Alexa Fluor 568 und Cy3) und HeNe 633nm (für Alexa Fluor 633, Cy5 und To-Pro-3) . </li><li> Verwenden sequenziellen Scan-Tool zu vermeiden oder zu vermindern das Übersprechen in dem alle Farbstoffe in Doppel-oder Dreifach-gefärbten Proben gleichzeitig begeistert sein. In der sequentiellen Scan-Modus, werden die Bilder in einer sequenziellen Reihenfolge aufgezeichnet werden. </li><li> Weitere allgemeine Informationen über Fluoreszenzfarbstoffe und Laser für die Anregung kann in &quot;Die konfokale Mikroskopie für Biologen&quot; gegründet werden, indem Hibbs (2004) </li></ol> 5. Repräsentative Ergebnisse Whole-mount Triple-Label konfokalen Mikroskopie in immnofluorescence Maus forelimb Haut an E15.5 mit Antikörpern gegen die pan-Endothelzell-Marker PECAM-1 (Abbildung 1A-C, D blau), die Neurofilament Marker 2H3 (Abbildung 1A grün) und die glatte Muskelzelle Marker αSMA (Abbildung 1A, B rot) zeigte eine charakteristische Verzweigungsmuster αSMA + Arterien, mit 2H3 + peripheren Nerven in den Gliedmaßen Haut ausgerichtet. Neben der Blutgefäße Verzweigungen, ist dieser Teil der Haut Gefäßsystem Modell zur Strukturierung von Lymphgefäßsystem Verzweigung mit Antikörpern gegen das lymphatische Endothel-Zell-Marker LYVE-1 (Abb. 1D, E rot) und Neuropilin2 (NRP2) (Abbildung 1D-, F grün-Studie ). <img src="/files/ftp_upload/2620/2620fig1.jpg" alt="Abbildung 1" /> Abbildung 1. (AC) Arterien align mit peripheren Nerven in der embryonalen Körpers Haut. Whole-mount Triple-Label konfokalen Mikroskopie immnofluorescence mit Antikörpern gegen die pan-Endothelzell-Marker PECAM-1 (AC, blau), die Neurofilament Marker 2H3 (A, grün) und der glatten Muskelzellen Marker αSMA (A und B, rot ) gezeigt. Am E15.5, 2H3 + peripheren Nerven (offene Pfeile) assoziieren mit Arterien (Pfeilspitzen), die durch αSMA abgedeckt werden + glatten Muskelzellen. (DF) Lymphgefäßsystems in der embryonalen Körpers Haut. Triple-Label konfokalen Mikroskopie immnofluorescence mit Antikörpern gegen die pan-Endothelzell-Marker PECAM-1 (D, blau), des lymphatischen Endothelzellen Marker LYVE-1 (D und E, rot) und Neuropilin2 (NRP2) (D und F, grün ) gezeigt. Lymphgefäße werden visualisiert sowohl LYVE-1 und NRP2, während LYVE-1 wird auch durch eine Teilmenge von Makrophagen (offene Pfeilspitzen) ausgedrückt. Maßstab: 100 um.

<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>

Das Gefäßsystem ist entscheidend für die Entwicklung der Organe während der Embryonalentwicklung sowie für Orgel Wartung und reproduktiven Funktionen in die Erwachsenen, weil es genügend Sauerstoff und Nährstoffe zu den Organen liefert. Proper Gefäßnetz ist mit komplexen und mehrstufigen Verfahren durch Angiogenese, in dem bereits bestehenden Netz von Kapillaren mit stark verzweigten und hierarchischen Strukturen neu organisiert ist gut etabliert. Obwohl zahlreiche Arbeiten haben gezeigt worden, dass eine Vielz...

Antibodies Pan-Endothelzell-Marker <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> PECAM-1 </td><td> Armenische Hamster (M) </td><td> Chemicon </td><td> MAB1398Z </td><td> 1:100-Verdünnung Nr. 1 </td></tr><tr><td> PECAM-1 </td><td> Rat (M) </td><td> BD </td><td> 553369 </td><td> 1:300 Verdünnung </td></tr><tr><td> VEGFR2 </td><td> Rat (M) </td><td> eBioscience </td><td> 14-5821-82 </td><td> 1:200-Verdünnung </td></tr><tr><td> CD34 </td><td> Rat (M) </td><td> eBioscience </td><td> 13-0341 </td><td> 1:300 Verdünnung </td></tr><tr><td> Collagen IV </td><td> Rabbit (P) </td><td> AbD Serotec </td><td> 2150-1470 </td><td> 1:300 Verdünnung Nr. 2 </td></tr></tbody></table> Arterielle Endothelzellen Marker <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> Neuropilin1 </td><td> Rabbit (P) </td><td> Der Alex Kolodkin Labor # 3 </td><td></td><td> 1:3000 Verdünnung </td></tr><tr><td> Unc5H2 </td><td> Goat (P) </td><td> R &amp; D </td><td> AF1006 </td><td> 1:200-Verdünnung </td></tr></tbody></table> Venöse Endothelzellen Marker <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> EphB4 </td><td> Goat (P) </td><td> R &amp; D </td><td> AF446 </td><td> 1:100-Verdünnung </td></tr></tbody></table> Lymphatische Endothelzellen Marker <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> LYVE-1 Nr. 4 </td><td> Rabbit (P) </td><td> Abcam </td><td> ab14917 </td><td> 1:200-Verdünnung </td></tr><tr><td> LYVE-1 Nr. 4 </td><td> Rat (M) </td><td> MBL </td><td> D225-3 </td><td> 1:200-Verdünnung </td></tr><tr><td> Prox-1 </td><td> Rabbit (P) </td><td> Chemicon </td><td> AB5475 </td><td> 1:1000-Verdünnung </td></tr><tr><td> Prox-1 </td><td> Goat (P) </td><td> R &amp; D </td><td> AF2727 </td><td> 1:50-Verdünnung </td></tr><tr><td> Neuropilin2 </td><td> Rabbit (P) </td><td> Cell Signaling </td><td> 3366 </td><td> 1; 100-Verdünnung </td></tr><tr><td> Podoplanin </td><td> Goldhamster (M) </td><td> Hybridoma der Bank </td><td> 8.1.1 </td><td> 1:200-Verdünnung </td></tr></tbody></table> Glatte Muskelzellen / Perizyten Marker <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> αSMA-Cy3 </td><td> Maus (M) # 5 </td><td> Sigma </td><td> c-6198 </td><td> 1:500-Verdünnung Nr. 6 </td></tr><tr><td> NG2 </td><td> Rabbit (P) </td><td> Chemicon </td><td> AB5320 </td><td> 1:200-Verdünnung </td></tr><tr><td> SM22α </td><td> Rabbit (P) </td><td> Abcam </td><td> ab14106 </td><td> 1:200-Verdünnung </td></tr></tbody></table> Antikörper für GFP-Reporter <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> GFP </td><td> Rabbit (P) </td><td> Invitrogen </td><td> A11122 </td><td> 1:300 Verdünnung </td></tr><tr><td> GFP </td><td> Rat (M) </td><td> Nacalai Tesque </td><td> 04404-84 </td><td> 1:1000-Verdünnung </td></tr><tr><td> GFP </td><td> Chick (P) </td><td> Chemicon </td><td> P42212 </td><td> 1:300 Verdünnung </td></tr></tbody></table> Antikörper für LacZ Reporter <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> β-gal </td><td> Rabbit (P) </td><td> MP Biomedical </td><td> 55976 </td><td> 1:5000 Verdünnung </td></tr><tr><td> β-gal </td><td> Goat (P) </td><td> AbD Serotec </td><td> 4600-1409 </td><td> 1:500 Verdünnung </td></tr><tr><td> β-gal </td><td> Chick (P) </td><td> Abcam </td><td> ab9361 </td><td> 1:200-Verdünnung </td></tr></tbody></table> Antikörper für periphere Axon <table border="1"><tbody><tr><td> Antikörper </td><td> Spezies </td><td> Firma </td><td> Katalog # </td><td> Arbeitsbedingung </td></tr><tr><td> 2H3 </td><td> Maus (M) # 7 </td><td> Hybridoma der Bank </td><td> 2H3 </td><td> 1:200-Verdünnung </td></tr><tr><td> Tuj1 </td><td> Maus (M) # 8 </td><td> Covance </td><td> MMS-435P </td><td> 1:500 Verdünnung </td></tr><tr><td> Peripherin </td><td> Rabbit (P) </td><td> Chemicon </td><td> AB1530 </td><td> 1:1000-Verdünnung </td></tr></tbody></table> Antikörper für die Migration von Schwann-Zellen <table border="1"><tbody><tr><td> BFABP </td><td> Rabbit (P) </td><td> Die Thomas Müller-Labor Nr. 9 </td><td></td><td> 1:3000 Verdünnung </td></tr></tbody></table> (P): polyklonaler Antikörper, (M): monoklonaler Antikörper # 1: Goat anti-armenischen Hamster-Cy3 (Jackson ImmunoResearch 127-165-160) Antikörper sollte als sekundärer Antikörper verwendet werden. # 2: Die Collagen IV Antikörper kann verwendet werden, um die Blutgefäße nach in-situ-Hybridisierung nachgewiesen werden. # 3: Die Neuropilin1 Antikörper wird freundlicherweise von der Alex Kolodkin Labor in der Johns Hopkins University zur Verfügung gestellt. Sheep anti-human Neuropilin1 Antikörper ist in R &amp; D (AF3870), obwohl wir es noch nicht getestet noch. # 4: Die LYVE-1-Antikörper auch erkennen, eine Teilmenge von Makrophagen in der embryonalen Haut. # 5: Die Anti-αSMA Antikörper Maus IgG2a monoklonaler Antikörper. # 6: Die Cy3-konjugierten αSMA Antikörper für 1 Stunde bei Raumtemperatur zusammen mit sec inkubiertondary Antikörper für andere primäre Antikörper. # 7: 2H3-Antikörper IgG1 monoklonaler Antikörper gegen Neurofilament. # 8: Tuj1 Antikörper Maus IgG2a monoklonaler Antikörper gegen Neuron-spezifische Klasse III beta-Tubulin. # 9: Die BFABP (Gehirn-spezifische fatty acid binding protein)-Antikörper wird freundlicherweise von der Thomas Müller-Labor in Max-Delbrück-Centrum für Molekulare Medizin zur Verfügung gestellt.

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

Whole-mount immunhistochemische Analyse zur Abbildung des gesamten Gefäßsystem ist von zentraler Bedeutung für das Verständnis der zellulären Mechanismen der Verzweigung Morphogenese. Wir haben das Bein Haut Gefäßsystem Modell der vaskulären Entwicklung, in der eine bereits bestehende primitive Kapillarplexus in eine hierarchisch verzweigten Gefäßnetz reorganisiert wird Studie entwickelt. Whole-mount konfokalen Mikroskopie mit mehreren Kennzeichnung ermöglicht robuste Bildgebung von intakten Blutgefäßen sowie deren zelluläre Komponenten, einschließlich Endothelzellen, Perizyten und glatte Muskelzellen, mit spezifischen fluoreszierenden Marker. Advances in diesem Teil der Haut Gefäßsystem Modell mit genetischen Studien haben ein besseres Verständnis molekularer Mechanismen der vaskulären Entwicklung und Strukturierung. Das Glied Haut Gefäßsystem Modell wurde verwendet, zu untersuchen, wie peripheren Nerven eine räumliche Vorlage liefern für die Differenzierung und Strukturierung von Arterien. Dieses Video Artikel beschreibt eine einfache und robuste Protokoll zu intakten Blutgefäßen mit vaskulären spezifische Antikörper und fluoreszierende Sekundärantikörper werden, die für vaskularisierte embryonale Organe, wo wir in der Lage sind, den Prozess der vaskulären Entwicklung folgen Fleck.

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

Wir stellen Ihnen eine ganze-mount Immunhistochemie und konfokaler mit mehreren Kennzeichnung für die Analyse von komplizierten Gefäßnetz Bildung in embryonalen Maus Gliedmaßen Haut.

Whole-mount Immunhistochemische Analyse für Embryonale Limb Haut Gefäßsystem: ein Modellsystem zur Vascular Branching Morphogenese in Embryo-Studie

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

whole-mount-immunohistochemical-analysis-for-embryonic-limb-skin

Research

JoVE Journal

Biology

17.5K Aufrufe.  National Heart, Lung, and Blood Institute, National Institutes of Health. Wir stellen Ihnen eine ganze-mount Immunhistochemie und konfokaler mit mehreren Kennzeichnung für die Analyse von komplizierten Gefäßnetz Bildung in embryonalen Maus Gliedmaßen Haut.

Serie: 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.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

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

Forschung

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.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

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.

Zebrafisch Whole Mount High-Resolution Doppel Fluorescent In-situ Hybridisierung

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.

Embryonalen Maus-Lung Kultur, A-System, die molekularen Mechanismen des Branching Bewerten

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 Einrichtung zur Rückgewinnung von kryokonservierten Proben versehentlich fallengelassen in Kryo-Lagerbehälter

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.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

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.

RNA<em&gt; In situ</em&gt; Hybridisierung in whole mount Embryonen und Histologie für Marine Elasmobranchs Angepasst

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.

Maus embryonalen Entwicklung in einem Serum-freien 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 ...

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.

Die Analyse der Apoptose in Zebrafischembryonen von Whole-mount Immunfluoreszenz an aktivierte Caspase 3 Detect

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