Diese Arbeit wurde von der National Science Foundation Grant IOS-0817658 unterstützt.

Der Zebrafisch Verfahren in diesem Protokoll wurden von der Institutional Animal Care und Use Committee (IACUC) an der Virginia Commonwealth University genehmigt. 1. Vorbereitung der Reagenzien <ol><li> 4% PFA / PBS. Wiegen 8 g Paraformaldehyd (PFA) in der Abzugshaube. Während noch in der Dunstabzugshaube, lösen sich die trockene PFA in ~ 80 ml ​​destilliertem H 2 O unter Rühren und Erwärmen auf 50 ° C. Unter Rühren hinzufügen, 3-10 Tropfen frischer 1 N NaOH bis PFA vollständig gelöst ist und Lösung klärt. Vom Herd nehmen und mit 100 ml 2x phosphatgepufferter Kochsalzlösung (PBS). Bringen Volumen auf 200 ml mit destilliertem H 2 O. Abkühlen auf RT und bestätigen pH-Wert von 7,4 vor dem Gebrauch. Lagerung im Dunkeln bei 4 ° C, aber verwenden innerhalb einer Woche. </li><li> Verwenden Sie 100% Methanol ohne Ergänzungen. Verwenden Sie 95% Ethanol ohne Ergänzungen. </li><li> Bereiten Phosphat-gepufferter Tween (PBT). Ergänzen phosphatgepufferter Kochsalzlösung (PBS) mit 0,1% Tween-20. In 0.5 ml einer 20% igen Tween-20-Stammlösung zu 100 ml 1x PBS. Lagern bei RT. </li><li> Bereiten phosphatgepufferter Triton-X (PBTx). Ergänzen PBS mit 0,1% Triton X-100. 0,5 ml einer 20% Triton X-100 Lager zu 100 ml PBS. Lagern bei RT. </li><li> Bereiten Sie 10% NGS / PBTx. Normales Ziegenserum (NGS) blockiert unspezifische Bindungsstellen. Shop NGS bei -20 ° C. So verwenden, 1 ml bis 9 ml PBTx. </li><li> Vorbereitung 50% Glycerin / PBS. Gründlich mischen zu gleichen Teilen aus 2x PBS und 100% Glycerin. Lagern bei RT. </li></ol> 2. Embryo Fixation <ol><li> . (. ZB β-Actin: CAAX-GFP) Abstammung Erhalten Wildtyp (AB und WIK) oder transgenen Embryos durch natürliche Paarungen. Falls gewünscht, injizieren Embryonen mit Konstrukten oder Morpholinos, wie beschrieben. 15,16 </li><li> Embryonen erhöhen. Sammeln Embryonen und Inkubation bei 28,5 ° C in Gegenwart von 0,003% 1-Phenyl-2-thioharnstoff (PTU) Pigmentierung zu blockieren, wie beschrieben. 17 </su<html> p&gt; </li><li> Fix. Wenn Embryonen den gewünschten Entwicklungsstadium erreicht haben, 18, &#160; betäuben die Embryonen mit MESAB, entfernen Sie so viel Wasser wie möglich System und fügen Sie frisch 4% PFA / PBS für 3-4 Stunden bei Raumtemperatur. HINWEIS: In Zeiten von weniger als 20 Stunden nach der Befruchtung (HPF), fix vor dechorionation. Manchmal nach 20 HPF, dechorionate vor der Fixierung mit zwei Paaren von feinen Spitze einer Pinzette unter einem Binokular. </li><li> Ändern Lösungen. Transfer-Embryonen eine breite Bohrung Pipette, wie beschrieben. 17 Lösungen wechseln in 1,5 ml Mikrozentrifugenröhrchen mit einer Kappe bedeckt, einfach, indem Embryonen durch die Schwerkraft ohne Zentrifugation zu begleichen. </li><li> Post-Fixierungs Nach 3 -. 4 Stunden, entfernen PFA / PBS, ersetzen mit 100% Methanol und bei -20 ° C für mindestens 48 Stunden. HINWEIS: Für maximale P-CaMK-II-Immunreaktivität, begrenzen Gesamt Methanol Lagerzeit bis zu einer Woche. </li></ol>&quot;&gt; 3. Die Immunfärbung Ganze Embryonen <ol><li> Setzen Sie ein Minimum von 10 Embryonen für jede Versuchsbedingung in verschlossenen 1,5 ml Mikrozentrifugenröhrchen, und beschriften. </li><li> Lassen Sie die Embryonen zu regeln. Entfernen und entsorgen Methanol und rehydratisieren mit progressiven wäscht mit 0,5 ml Ethanol-Konzentrationen abnimmt, wie im nächsten Schritt angegeben. Jeder Schritt ist ein 5 min Waschen mit Schaukel. </li><li> Progressiv mit diesen 5 Lösungen rehydriert: 66% Ethanol / 33% PBTx, 33% Ethanol / 66% PBTx 100% PBTx 100% PBTx (dies ist die erste Wiederholung PBTx), 100% PBTx (dies ist die zweite Wiederholung von PBTx). </li><li> Entfernen letzten PBTx waschen. 0,5 ml 10% NGS / PBTx. mindestens 1 Stunde bei RT inkubieren unter leichtem Schütteln und dann entfernen und Lösung verwerfen. </li><li> Bereiten primären Antikörperlösung durch in 10% NGS / PBTx verdünnen. Wenn Co-Immunfärbung verwenden höhere Affinität Antikörper zuerst. 500: mit der Maus anti-acetylierten Antikörper Tubulin monoklonalen verdünnt 1 Für diese Studie inkubiert. für example, wenn 10 Proben sind, kombinieren 6 ul der Stammantikörperlösung mit 3 ml 10% NGS / PBTx und dann 0,3 ml davon in jedes Röhrchen verteilt. </li><li> Hinzufügen 0,2-0,5 ml der verdünnten primären Antikörpers zu jedem Röhrchen alle Embryonen einzutauchen. Inkubieren unter leichtem O / N bei RT zu schaukeln. </li><li> Am Morgen, zu entfernen und Antikörperlösung zu verwerfen. 0,5 ml 2% NGS / PBTx überschüssigen primären Antikörpers weg zu waschen. Sanft für 5 min schaukeln. Entfernen und entsorgen Lösung. Zweimal wiederholen. </li><li> Dim Lampen oder verdünnen Sie den entsprechenden fluoreszenz konjugierten sekundären Antikörper in 10% NGS / PBTx so dass jeder Zustand mindestens 0,5 ml enthält. Mit der Maus-Anti-acetyliertes Tubulin primärer Antikörper, verwenden Sie die grün-fluoreszierenden Farbstoff Ziegen-Anti-Maus-IgG bei einer 1: 500-Verdünnung. </li><li> Inkubieren sekundären Antikörper für 4 Stunden unter leichtem im Dunkeln bei RT Schaukel. Von nun an Verfahren die Proben unter gedämpftem Licht und in einem dunklen Behälter brüten oder in Alufolie wickeln. </li><li>Am Ende der Inkubation entfernt und die sekundäre Antikörperlösung verwerfen. 0,5 ml 2% NGS / PBTx zu waschen. Sanft für 5 min schaukeln. Entfernen und entsorgen Lösung. Zweimal wiederholen. </li><li> Wenn Co-Immunfärbung, erhalten den zweiten primären Antikörper von einer anderen Spezies als der ersten primären Antikörper. In diesen Studien, folgen Sie dem Kaninchen-Anti-phospho CaMK-II-Antikörper von der rot-fluoreszierenden Farbstoff-konjugierten Ziegen-Anti-Kaninchen-IgG-Sekundärantikörpers. </li><li> Verdünnen Kaninchen-Anti-phospho CaMK-II-Antikörper 1.20. Für jedes Röhrchen von Embryonen, in 0,3 ml 10% NGS / PBTx aussetzen, dann 15 ul der Stammantikörperlösung hinzuzufügen. </li><li> Stellen Sie sicher, dass Embryonen werden eingetaucht und Lichter gedimmt werden. Inkubieren unter leichtem O / N bei RT im Dunkeln zu schaukeln. </li><li> Am Morgen unter der gedämpften Beleuchtung entfernen und Antikörperlösung zu verwerfen. 0,5 ml 2% NGS / PBTx. Sanft für 5 min schaukeln. Entfernen und entsorgen Lösung. Zweimal wiederholen. </li><li> Halten Overhead Lichter dimmen und verdünnen genug von dem entsprechenden fluoreszenz conjugated sekundären Antikörper, so dass jeder Zustand mindestens 0,5 ml. In dieser Studie wurden die verdünnten roten Fluoreszenzfarbstoff-konjugiertes Ziege-anti-Kaninchen-IgG-Sekundärantikörper (roter Kanal) 1: 500 in 10% NGS / PBTx. </li><li> Inkubieren zweiten sekundären Antikörper für 4 Stunden mit sanften im Dunkeln bei RT zu schaukeln. </li><li> Am Ende dieser Inkubation und unter der gedämpften Beleuchtung ist zu entfernen und die sekundäre Antikörperlösung zu verwerfen. 0,5 ml PBTx. Sanft für 5 min schaukeln. Entfernen und entsorgen Lösung. Zweimal wiederholen. Speicher in entweder PBTx oder 50% Glycerin / PBS in Abhängigkeit von dem Bildgebungsverfahren. </li></ol> 4. Die konfokale Fassung und -verarbeitung <ol><li> Berg Embryonen für die Bildgebung. Zeigen 1-5 Embryonen auf einem Glasträger. Erstellen einer Kammer zwischen dem Hauptdeckglas und dem Objektträger unter Verwendung von Deckglas-Fragmente auf jeder Seite der Kammer. Typischerweise werden vier # 1 Deckgläser verwendet, um diese Abstandshalter Stapel zu machen, ohne zu verkleben. HINWEIS: Keine Montage Medium notwendig. </li><li> Verwenden Sie eine 100X ÖlimmersionZiel einzigen Embryo in den Fokus Durchlicht zu bringen. Schalten Sie Licht. Schalten Sie konfokalen Mikroskop. Stellen Sie sicher, dass geeignete Laser (grün und / oder rot) eingeschaltet sind und Remote-Fokus Mithelfer eingreift. </li><li> Schalten Sie den konfokalen Programm. In der &quot;Acquire Bar&quot;, die richtige Ziel wählen. In der &quot;XY Basic&quot; Bar, setzen die Bildgröße auf 1.024 von den 1.024 Button. </li><li> In der &quot;Laser und Detektor&quot; Leiste klicken Sie auf den roten 488-Box und dem grünen 568 Feld Laser und Detektor für jeden Kanal zu schalten. Stellen Pinhole bis mittelgroß und passen Sie die Verstärkung in der &quot;Gain&quot; Bar jeder Kanal zu visualisieren. </li><li> Im &quot;Einstellungen Acquire&quot; Leiste auf &quot;Live&quot; Erfassung von Bildern zu beginnen. In der &quot;Einstellungen anzeigen&quot; Bar, deaktivieren Sie die &quot;Force Integral Zoom&quot; Box-Center in der &quot;Live&quot; -Fenster. </li><li> In der &quot;Einstellungen abrufen&quot; bar unter dem &quot;Z&quot; Registerkarte Schritt durch die Schichten des Bildes. Nach der Auswahl der number optischer Schnitte in das Bild zu übernehmen, in der &quot;Mitte&quot; der z-Ebene bis zu einem gewissen Punkt. </li><li> Im Rahmen der &quot;Z&quot;, klicken Sie das kleine rote &quot;Reference&quot; ein. Dadurch wird die RFA an dem Punkt Null, wenn die &quot;Mitte&quot; gewählt. Im Feld &quot;Schrittweite&quot; bezeichnet, wählen Sie die Dicke der Schichten (zwischen 0,25 &amp; mgr; m und 2,0 &amp; mgr; m ist ideal). Achten Sie auf das &quot;File Size&quot; ein und versuchen zu 1 GB zu begrenzen. HINWEIS: In der Regel bis zu 40 optische Schnitte von 0,5 bis 1,0 &amp; mgr; m erhalten werden, aber die Anzahl der Abschnitte können empirisch bestimmt werden. </li><li> Um die oben extreme des Bildes zu finden, klicken Sie auf den Kreis neben &quot;Top&quot; und bewegen sich durch die z-Schichten. Jetzt den Kreis klicken Sie neben &quot;Unten&quot;, nimmt der Computer das Bild wieder in der Schicht als Zentrum. bewegen wieder durch die Schichten der unteren extremen des Bildes zu finden. </li><li> Klicken Sie auf &quot;Live&quot; wieder in den &quot;Einstellungen abrufen&quot; Feld des Lasers zu stoppenErwerb. In diesem Fenster klicken Sie auf die roten Kisten etikettiert Durchschnitt und Z-Stapel. Diese Boxen wird grün. </li><li> In der &quot;Einstellungen abrufen&quot; ein, klicken Sie auf &quot;Single&quot; das ganze Reihe von Bildern zu erwerben. Sie können den Fortschritt überwachen, da es in beiden Kanälen durchsucht und durch die gesamte Z-Stapel. </li><li> Zu speichern, klicken Sie auf das Fenstervolumen und klicken Sie auf &quot;Speichern unter&quot; und den Dateinamen. Speichern als &quot;.ids&quot; -Datei. Um die Lautstärke zu machen, die Datei während der Z-Stapel noch geöffnet ist, wählen Sie das Menü Daten nach unten ziehen und klicken Sie auf &quot;Volumen machen&quot;. Speichern Sie die gerenderte Datei als TIFF-Datei. Dies ist das projizierte Bild, das in dieser Veröffentlichung dargestellt sind. </li></ol>

<ol>
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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Die PFA / Methanol-Verfahren wurde in diesem Labor mit dem vorrangigen Ziel der Optimierung der Immun des Phospho-T 287 -CaMK-II-Epitop während Zebrabärblingentwicklung entwickelt. Diese Methode erfolgreich P-CaMK-II bei der Bildung von mehreren ciliated Organe, einschließlich Zebrafisch KV, 12 Innenohr 14 und Niere. 13 besonders am KV Stufe diese Technik notwendig war, lokalisiert. Der Erfolg dieses Verfahrens ist wahrscheinlich auf eine Kombination von a) Minimierung von ...

The techniques described here are the outcome of studies that have sought to define downstream targets of Ca2+ signals during events that occur during early development, including fertilization, gastrulation, somitogenesis and trunk, eye, brain and organ formation.1-3 The original discoveries of embryonic Ca2+ signaling were dependent on the use of natural and engineered Ca2+ indicators, such as aequorin4 and fura-2.5 Even with current technology, the detection of transient elevations of Ca2+ requires cumbersome analytical tools and does not reveal the targets of such Ca2+ signals. 
This laboratory investigates Ca2+ signals that act through the Ca2+/calmodulin-dependent (multifunctional) protein kinase known as CaMK-II, an enzyme that is enriched in the central nervous system and originally identified as a regulator of long-term potentiation.6 CaMK-II is not brain-specific, is widely expressed and highly conserved throughout the entire lifespan and bodies of species throughout the animal kingdom, including invertebrates.7,8 CaMK-II has the unique capability of sustaining its own activity even after Ca2+ levels have diminished due to its ability to autophosphorylate at Thr287. In this autophosphorylated state, CaMK-II remains active in a Ca2+/CaM-independent manner, until dephosphorylated.6 Thus, the localization of phosphorylated CaMK-II (Thr287) can identify cells in which natural, relevant Ca2+ elevations have occurred. 
An antibody against autophosphorylated (P-Thr287) mammalian CaMK-II has been well-characterized and was initially used to localize activated CaMK-II in brain tissue.9 Zebrafish (Danio rerio) have seven CaMK-II genes10,11 whose protein products contain a sequence of MHRQE[pT287]VECLK in this region.10,11 This sequence is very similar to the phosphopeptide antigen used to create this rabbit polyclonal antibody (MHRQE[pT]VDCLK; Upstate/Millipore) and therefore it was not a complete surprise that this antibody cross-reacted with zebrafish CaMK-II. This laboratory showed that this antibody reacts with zebrafish CaMK-II in proportion to autophosphorylation and Ca2+/CaM-independent activity.12 Additional pan-specific CaMK-II antibodies have also been shown to cross-react with zebrafish CaMK-II.13
This antibody has been used to demonstrate that zebrafish CaMK-II is preferentially activated in cells on one side of the zebrafish Kupffer's Vesicle (KV), the ciliated organ necessary for establishment of left/right asymmetry.12 This antibody was used to demonstrate that CaMK-II is transiently activated in four adjacent cells on the left side of the KV during the exact same developmental phase that organ positioning is determined.12 In addition to the Kupffer's Vesicle (KV), autophosphorylated (P-T287) was also located in specific intracellular sites in other ciliated tissues including the kidney, neuromasts, and inner ear.12,13 In the zebrafish kidney, P-T287-CaMK-II is enriched along the apical border of ciliated ductal cells and within cloacal cilia where it influences their assembly.13 Finally, in the developing inner ear, P-T287-CaMK-II is intensely concentrated at the base of cilia and influences cell differentiation through the Delta-Notch signal pathway.14 In summary, the detection of activated CaMK-II has pinpointed sites of intracellular Ca2+ release and illuminated potential new signaling pathways.
These discoveries were completely dependent on developing a sensitive and accurate method to localize activated (P-T287-autophosphorylated) CaMK-II. The methods to fix and immunostain the zebrafish KV, kidney and inner ear are described. The limitations of this technique are also described. These techniques should be useful to any investigator who seeks to obtain high-resolution images in two fluorescent channels of not just phospho-epitopes, but any epitope, during early vertebrate development.

<table><tbody><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma</td><td>P-7629</td><td>0.12% Stock solution. Dilute 1:40 in system water</td></tr><tr><td>Alexa488 anti-mouse IgG</td><td>Life Technologies</td><td>A11001</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 anti-rabbit IgG</td><td>Life Technologies</td><td>A11008</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa488 phalloidin</td><td>Life Technologies</td><td>A12379</td><td>Preferentially binds to F-actin</td></tr><tr><td>Alexa568 anti-mouse IgG</td><td>Life Technologies</td><td>A11004</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>Alexa568 anti-rabbit IgG</td><td>Life Technologies</td><td>A11011</td><td>Goat polyclonal, use at 1:500</td></tr><tr><td>anti-acetylated &amp;#945;-tubulin</td><td>Sigma</td><td>T7451</td><td>Mouse monoclonal, use at 1:500</td></tr><tr><td>anti-phospho-T&lt;sup&gt;287&lt;/sup&gt; CaMK-II</td><td>EMD Millipore</td><td>06-881</td><td>Rabbit polyclonal, use at 1:20</td></tr><tr><td>anti-total CaMK-II</td><td>BD Biosciences</td><td>611292</td><td>Mouse monoclonal, use at 1:20</td></tr><tr><td>Ethanol</td><td>Fisher</td><td>S96857</td><td>Lab grade, 95% denatured</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Glass coverslips</td><td>VWR</td><td>16004-330</td><td>#1&amp;nbsp; thickness</td></tr><tr><td>Glass microscope slides</td><td>Fisher</td><td>12-550-15</td><td>Standard glass slides</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A411</td><td>Store in freezer</td></tr><tr><td>Microcentrifuge tubes</td><td>VWR</td><td>20170-038</td><td>capped tubes, not sterile</td></tr><tr><td>Normal goat serum</td><td>Life Technologies</td><td>16210-064</td><td>Aliquot 1 ml tubes, store in freezer</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P-6148</td><td>Reagent grade, crystalline</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Quality Biological</td><td>119-069-131</td><td>10x stock solution or made in lab</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>BP-151</td><td>10% solution in water, store at RT</td></tr><tr><td>Tween-20</td><td>Life Technologies</td><td>85113</td><td>10% solution in water, store at RT</td></tr><tr><td>Compound microscope</td><td>Nikon</td><td>E-600</td><td>Mount on vibration-free table</td></tr><tr><td>C1 Plus two-laser scanning confocal</td><td>Nikon</td><td>C1 Plus</td><td>Run by EZ-C1 program, but upgrades use &quot;Elements&quot;</td></tr></tbody></table>

immunostaining phospho-epitopes in ciliated organs of whole mount zebrafish embryos

Die schnelle Proliferation von Zellen, die gewebespezifische Expression von Genen und die Entstehung von Signalisierungsnetze charakterisieren frühen embryonalen Entwicklung aller Wirbeltiere. Die Kinetik und die Lage der Signale - auch innerhalb einzelner Zellen - in den sich entwickelnden Embryo ergänzt die Identifizierung wichtiger Entwicklungsgene. Immunfärbung Techniken beschrieben, die die Kinetik der intrazellulären und ganzen Tier Signalen in Strukturen so klein wie primäre Zilien definieren abgebildet wurden. Die Techniken zur Fixierung, Imaging und Bearbeitung von Bildern mit einem Laser-Scanning-konfokalen Mikroskops verwendet, kann in nur 36 Stunden abgeschlossen sein. Zebrafisch (Danio rerio) ist eine wünschenswerte Organismus für Forscher, die versuchen, Studien in einem Wirbeltierarten durchzuführen, die erschwinglich und relevant für die menschliche Krankheit. Genetic Knockouts oder Niederschlägen muss durch den Verlust der tatsächlichen Proteinprodukt bestätigt. Eine solche Bestätigung von Proteinverlustwerden können unter Verwendung der hier beschriebenen Techniken erreicht. Clues der Signalwege kann auch durch Verwendung von Antikörpern entschlüsselt werden, der mit Proteinen reaktiv sind, die durch Phosphorylierung posttranslational modifiziert wurden. Die Erhaltung und die phosphorylierten Zustand eines Epitops Optimierung ist daher entscheidend für diese Bestimmung und wird von diesem Protokoll erreicht. Diese Studie beschreibt Techniken Embryonen während der ersten 72 Stunden der Entwicklung und zu beheben eine Vielzahl von relevanten Epitope mit Zilien in das Vesikel (KV), die Nieren und das Innenohr des Kupffer co-lokalisieren. Diese Verfahren sind einfach, nicht Dissektion erforderlich und kann in einer relativ kurzen Zeitspanne abgeschlossen werden. Projizieren konfokale Bildstapel zu einem einzigen Bild ist ein nützliches Mittel, um diese Daten zu präsentieren.

Optimale Bedingungen für die Visualisierung von Phospho-Epitope Methoden, um die Immun von Protein-Epitope in Zebrafischembryonen beschrieben haben relativ spärlich gewesen im Vergleich zu denen für mRNAs über in-situ-Hybridisierung zu lokalisieren. Fixiermittel bei der Lokalisierung von Protein-Epitope in Geißelzellen des Zebrafischembryonen verwendet haben 4% PFA / PBS und Dent Fixierungsmittel...

Watch this Scientific Journal Video about Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos at JoVE.com

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Es sind Techniken beschrieben Phospho-Epitope in ganz Zebrafischembryonen zu Immunfärbung und dann leiten zweifarbige Leuchtstoff konfokalen Lokalisierung in zellularen Strukturen so klein wie primäre Zilien. Die Techniken zur Fixierung und Bildgebung können die Position und die Kinetik des Erscheinens oder Aktivierung spezifischer Proteine ​​definieren.

Immunfärbung Phospho-Epitope in Ciliated Organe Ganze Berge Zebrafischembryonen

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

immunostaining-phospho-epitopes-ciliated-organs-whole-mount-zebrafish

Research

JoVE Journal

Chemistry

8.0K Aufrufe.  Virginia Commonwealth University.  Es sind Techniken beschrieben Phospho-Epitope in ganz Zebrafischembryonen zu Immunfärbung und dann leiten zweifarbige Leuchtstoff konfokalen Lokalisierung in zellularen Strukturen so klein wie primäre Zilien. Die Techniken zur Fixierung und Bildgebung können die Position und die Kinetik des Erscheinens oder Aktivierung spezifischer Proteine ​​definieren. 

Serie: Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

High Precision Zinc Isotopic Measurements Applied to Mouse Organs

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn and 70Zn) which are naturally fractionated between mouse organs. We first show how to dissolve the different organs in order to free the Zn atoms; this step is realized by a mixture of HNO3 and H2O2. We then purify the zinc atoms from all the other elements, in particular from isobaric interferences (e.g., Ni), by anion-exchange chromatography in a dilute HBr/HNO3 medium. These first two steps are performed in a clean laboratory using high purity chemicals. Finally, the isotope ratios are measured by using a multi-collector inductively-coupled-plasma mass-spectrometer, in low resolution. The samples are injected using a spray chamber and the isotopic fractionation induced by the mass-spectrometer is corrected by comparing the ratio of the samples to the ratio of a standard (standard bracketing technique).&#160;This full typical&#160;procedure produces an isotope ratio with a 50 ppm (2 s.d.) reproducibility.

We present the technique to measure with high precision zinc isotope ratios in mouse organs.

High Precision Zink Isotopenmessungen an Maus-Orgeln Angewandte

We present a procedure to measure with high precision zinc isotope ratios in mouse organs. Zinc is composed of 5 stable isotopes (64Zn, 66Zn, 67Zn, 68Zn ...

Forschung

Chemie

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Radiomarkierung und Quantifizierung von Cellular Ebenen Phosphoinositide von High Performance Liquid Chromatography-gekoppelten Durchflussscintilla

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualisierung von Multiciliated Zellen in der Zebrabärbling durch ein kombiniertes Protokoll der ganze Berg Leuchtstofflampen In Situ Hybridisierung und Immunfluoreszenz

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Entwicklungsbiologie

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 ...

Biologie

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Ganzer Berg Immunhistochemie in Zebrafisch Embryonen und Larven

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Single Cell Fate Mapping im Zebrafisch

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunfärbung von Dissected Zebrafisch embryonale Herz

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

Ein effizientes Protokoll zur Beurteilung der Modulation der ERK-Aktivität in frühen Modellen des Zebrafisch-Noonan-Syndroms mittels Live-FRET-Mikroskopie und Immunfluoreszenz

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Biochemie

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

In situ Hybridisierung kombiniert mit Immunhistochemie in kryogeschnittenen Zebrafischembryonen

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

Neurowissenschaften

Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish embryo is an excellent tool for examining such interactions with an in vivo model. Whole-mount immunohistochemical and immunofluorescence assays are frequently applied in zebrafish embryos to assess protein expression. However, it can be difficult to achieve accurate mapping of co-localized proteins in three-dimensional space. In addition, some studies may require the use of two antibodies that are not compatible with the same technique (e.g., antibody 1 is only suitable for immunohistochemistry and antibody 2 is only suitable for immunofluorescence). The purpose of the method described herein is to perform sequential immunofluorescence and/or immunohistochemistry on individual cryosections derived from early-stage zebrafish embryos. Here we describe the use of sequential rounds of immunofluorescence, imaging, immunohistochemistry, imaging for a single cryosection in order to achieve precise identification of protein expression at the single-cell level. This methodology is suitable for any study in early-stage zebrafish embryos that requires accurate identification of multiple protein targets in individual cells.

This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from early-stage zebrafish embryos enabling precise colocalization analyses in specific cell populations.

Sequenzielle Immunfluoreszenz und Immunhistochemie an kryosektionierten Zebrafisch-Embryonen

Investigation of intercellular interactions often requires discrete labeling of specific cell populations and precise protein localization. The zebrafish ...