Dank Marie Birne, Areti Tsiola, Jaymie Estevez und Arelys Uribe. Diese Arbeit wurde teilweise durch CUNY Research Foundation und Howard Hughes Medical Institute Stipendium Summer Program für Undergraduate Students (SPUR) und NIH RO3 41702-00-01 unterstützt. Forschung wurde auch von Queens College und einige der Experimente wurden an Geräten aus der Core Facility for Imaging, Cellular and Molecular Biology am Queens College getan unterstützt.

Zebrafisch erhoben wurden mit Standardprotokollen 13 in der Queens College Tierhaltung bei 28 ° C. Individuelle Fische paarweise oder in Gruppen Paarungen sollten vorgeformt und die daraus resultierenden Embryonen gereinigt und in einem 28 ° C Inkubator. Die Gesundheit der Fische sollten täglich überprüft werden. Bei 5-Tage nach der Befruchtung (DPF), sollten die Fische in Tanks bei einer 10-15 Fischdichte getrennt werden und in die Fisch-Anlage Kinderzimmer. 15, 30, 45, 60, 90, 180 dpf: Für das Protokoll hier vorgestellten Zebrafisch wurden Larve durch adulte Stadien gesammelt. 1. Zebrafisch Thema Sammlung und Fixierung <ol><li> Entfernen Sie Fisch aus Lebensraum Tank mit Netz und in 0,2% Tricaine zu betäuben. </li><li> Legen Sie narkotisierten Fisch in Eiswasser für 15 Minuten bis einschläfern. </li><li> Machen Sie frischen 4% Paraformaldehyd (PFA) in 1x Phosphat-gepufferter Kochsalzlösung (PBS). </li><li> Inkubieren gesammelt Fisch in 4% PFA für 1-2 Stunden bei RT über Nacht bei 4 ° C in einer Petrischale in Parafilm umwickelt. Das hält die Fische flach und macht später Dissektion viel einfacher. </li><li> Nach der Fixierung zweimal in PBS gründlich mit Tween (PBT). Fische können in PBT werden halten bei 4 ° C für bis zu 10 Tage. </li></ol> 2. Mess-Fisch und Dissektion <ol><li> Legen Sie einzelne Fische in Petrischale zur Hälfte mit frischem PBS gefüllt und legen Sie es auf der rechten Seite. </li><li> Messen Sie die Länge der Fische von der Schnauze bis Rutenansatz (die Schwanzflosse), ohne Heckflossen, mit einem digitalen Messschieber mm (Abbildung 1). Dies ist die Standard-Länge (SL). </li><li> Jeder Fisch kann eine Zahl / Buchstaben-Kombination zu einem einzelnen Fisch und die daraus resultierenden seziert Herz für die spätere Analyse verfolgt werden können zugeordnet werden. Daten, einschließlich der elterlichen Lager Anzahl, Alter bei Fixierung, SL-Messungen wurden in ein Tabellenkalkulationsprogramm eingegeben und organisiert auf diesen Etiketten basiert. </li><li> Vor Dissektion Label PCR-Röhrchen-Streifen mit Tracking-Nummer zugeordnet und mit PBT-oder andere angemessene Lösung für die weitere Analyse zu füllen. </li><li> Richten Sie den Fisch Bauchseite bis in Petrischale, während die Stabilisierung des Körpers mit einer Pinzette hält den Kopf zwischen den Augen und Kiemen (Abbildung 2). </li></ol> Kleinere Fische (kleiner als 12mm SL): <ol start="4"><li> a) Mit scharfen Pinzette oder einer Mikro-Nadel in einem Stifthalter, entfernen Sie die Brustmuskeln und Flossen aus dem Körper zum Herzen zu offenbaren. Die konstante Bewegung der Zange beim Präparieren kann Ströme im PBT, dass die Fische zu bewegen. Dies ist besonders problematisch für kleinere Fische, also ein Mikro-Nadel kann verwendet werden, um Brustmuskeln und Flossen entfernen und öffnen Sie die Körperhöhle werden. </li><li> a) Sie vorsichtig mit der Pinzette in das Herz Schaufel aus dem Hohlraum unter dem Atrium. Alternativ in ein paar Fische können Sie möglicherweise das Herz, indem Sie vorsichtig die bauchige aus und der Rest des Herzens folgen zu entfernen. Achten Sie auf die Lage des Atriums, wenn mit dieser Technik, da es leicht beschädigt werden können. Wenn es sich um zusätzliche Gewebe, das mit dem Herzen, das ist in Ordnung und kann nach geräumt werden wird. </li><li> a) Wenn der Fisch enthält einen fluoreszierenden kardiale Marker wie Tg (myl7: GFP) twu34, verwenden fluoreszierende Spielraum für Zerlegung und überprüfen Entfernen des Herzens durch Fluoreszenz (Abbildung 3). </li><li> a) Wenn das Herz aus dem Hohlraum entfernt ist, verwenden Sie die Zange, um das Herz und ein Mikro-Nadel halten, um zusätzliche nicht-kardialen Gewebe und der epikardialen Futter aus dem außerhalb des Herzens zu entfernen. </li></ol> Größere Fische (größer als 12 mm SL): <ol start="4"><li> b) Mit Feder behandelt Mikro-Schere, machen drei Schnitte von weniger als 1mm tief: 1 - Schnitt quer durch die Kiemen 2 - Schnitt quer am vorderen Bauch 3 - sagittal geschnitten auf Bauchseite verbindet die beiden Schnitte (Abbildung 4). </li><li> b) Mit scharfen Pinzette entfernen Sie die Brustmuskeln und Flossen aus dem Körper in die Leibeshöhle öffnen und zeigen die silbrigen Gewebe des Perikards (Abbildung 5A). Entfernen Sie diese Perikardgewebe und das Herz wird sichtbar (Abbildung 5B). </li><li> b) Mit der Mikro-Schere, um die Arterie mit dem bauchigen arteriosus, befindet sich über dem Herzen schneiden. Sobald diese Arterie zu senken, das Pinzette Tipps unter dem Atrium, verwenden Sie die Zange, um das Herz Schaufel aus dem Hohlraum. Wenn es sich um zusätzliche Gewebe, das mit dem Herzen, das ist in Ordnung und kann später entfernt werden wird. </li><li> b) Wenn das Herz aus dem Hohlraum entfernt ist, werden die zwei Zangen an das Herz zu halten und zu entfernen Extra-Gewebe oder eine Pinzette, um das Herz zu halten und ein Mikro-Nadel, um zusätzliche, nicht-kardialen Gewebe zu entfernen. Die epikardialen Futter, offensichtlich durch seine schwarze Pigmentierung, kann von der Außenseite des Herzens entfernt werden, wenn durch vorsichtiges Schaben das Gewebe gewünscht. </li></ol> 3. Fotografie der Herzen <ol><li> Bereiten Sie eine 23g / L Nähragar Petrischale und decken erstarrten Agar mit PBS. </li><li> Mit der Zange, eine Vertiefung in den Agar, dass das Herz in die gewünschte Ausrichtung für Aufnahmen Rest ermöglicht. </li><li> Remove des Herzens aus dem PCR-Röhrchen, indem die Spitze des bauchigen mit der Zange fest. Legen Sie das Herz in die Agar-Schale. </li><li> Orient im Herzen in Agar zu fotografieren. Die Probe kann gedreht werden, nach jedem Bild, um alle möglichen Orientierungen (Abbildung 6). </li><li> Verwenden Sie Oberlicht und kurze Belichtungszeit, um die Herzen zu fotografieren. Nehmen Sie die Einstellung für die Lichtqualität mit der Exposition als notwendig, um die beste Aufnahme zu erhalten. </li></ol> 4. Repräsentative Ergebnisse: Ein Beispiel für einen gut präparierten reines Herz ist in mehreren Ausrichtungen in Abbildung 6 dargestellt. Jedes Herz wird einige Unterschiede in der Morphologie insgesamt, aber das Herz sollte vollständig sein, mit einer intakten Ventrikel, Atrium und bauchige arteriosus. Einige Beispiele für diese Variante in insgesamt Morphologie sind in Abbildung 7 gezeigt. Die schwarz getupften epikardialen Gewebe kann auch aus dem Herzen entfernt. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Messen Sie den Fisch aus der Schnauze bis zum Schwanzstiel mit einem digitalen mm Dicke. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Halten Sie den Fisch mit einer Zange zwischen den Augen und Kiemen. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Abbildung 3"/> Abbildung 3 A kardiale spezifische fluoreszierende Marker, wie transgenen Tg (myl7: GFP). Twu34, hier gezeigt, kann bei der Visualisierung Herzen von kleineren Fischen seziert Hilfe. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Abbildung 4"/> Abbildung 4 Drei Einschnitte, die Brustmuskeln und die Haut entfernen: 1 - Schnitt quer durch die Kiemen 2 - Schnitt quer am vorderen Bauch 3 - sagittal geschnitten auf Bauchseite verbindet die beiden Schnitte.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Abbildung 5"/> . Abbildung 5 Black Briefe beschriften Sie die Kammern des Herzens: bauchige arteriosus (ba), Ventrikel (v), und Atrium (a). A-Das obere Leibeshöhle ist offen für den silbrigen Gewebe des Herzbeutels zeigen. B-Nach dem Entfernen der Herzbeutel, das Herz ist sichtbar und leicht zu entfernen. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Abbildung 6"/> Abbildung 6. Unterschiedliche Orientierungen des Herzens gezeigt. Schwarze Buchstaben beschriften Sie die Kammern des Herzens: bauchige arteriosus (ba), Ventrikel (v), und Atrium (a). Graue Pfeile zeigen Lage und Orientierung des Herzens in Bezug auf seine normale Lage im Körper: anterior (A), posterior (P), dorsal (D), ventral (V), links (L) und rechte (R). Maßstabsbalken zeigt 400μm. A-ventral des Herzens B-Dorsalansicht des Herzens C-Seitenansicht von der linken Seite D-Seitenansicht von der rechten Seite <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Abbildung 7"/> Abbildung 7. Morphologie der Herzen in verschiedenen Größen dargestellt. Schwarze Buchstaben beschriften Sie die Kammern des Herzens: bauchige arteriosus (ba), Ventrikel (v), und Atrium (a). Skala bar angegeben 400 um. AC-Hearts Form Fisch mit SL weniger als 12mm DF-Hearts Form Fisch mit SL größer als 12mm

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T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

Keine Interessenskonflikte erklärt.

Diese Methoden zur Präparation des Zebrafisch-Herzen post-embryonale Fisch ermöglicht die vollständige Entfernung von einem unbeschädigten Herzen. Während diese Technik ist unkompliziert und bietet Führungen für externe Schnitte, die eine Beschädigung des Herzens wird, ist eine ruhige Hand unerlässlich für kleinere Fische. Ein wichtiger Schritt bei der Präparation ist es, die silbrig Herzbeutel zu identifizieren, wie das Herz befindet sich direkt in diesem Sack. Visualisierung der...

<table border="1"><tbody><tr><th> Material </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Paraformaldehyd </td><td> EMD </td><td> PX0055 </td><td> .04g/mL In PBS </td></tr><tr><td> Zange Nr. 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> Micro-Schere </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> Pin Halter </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> Micro-Nadeln </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> Nähragar </td><td> BBL </td><td> 11472 </td><td> 23g / L in destilliertem H 2 O </td></tr><tr><td> Petrischalen </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR 8-Tube-Strips </td><td> USA Scientific </td><td> 1402-2500 </td><td></td></tr><tr><td> Digitaler Messschieber </td><td> Fisher Scientific </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Zebrafische haben sich zu einem nützlichen und praktischen Modell-Organismus für die Untersuchung der embryonalen Entwicklung des Herzens (siehe letzter Zeit 1-6), jedoch arbeiten die Prüfung post-embryonale durch erwachsene Herzentwicklung wurde 7-10 beschränkt. Die Untersuchung der Veränderung der Morphologie der Reifung und Alterung Herzen sind durch den Mangel an verfügbaren Techniken für die Inszenierung und das Isolieren jugendlichen und erwachsenen Herzen eingeschränkt. Um die Entwicklung des Herzens über die Fische die Lebensdauer zu analysieren, sezieren wir Zebrafisch Herzen auf zahlreichen Bühnen und fotografieren sie für die weitere Analyse 11. Die morphologischen Eigenschaften des Herzens lassen sich leicht quantifizieren und einzelnen Herzen kann durch eine Vielzahl von Standard-Methoden analysiert werden. Zebrafische zu variablen Zinssätzen wachsen und Reifung korreliert besser mit der Größe der Fische als das Alter, also nach der Fixierung, wir fotografieren und messen Fisch Länge als ein Maß für Fisch Reifung. Dieses Protokoll erklärt zwei unterschiedliche, Größe abhängig Dissektion Techniken für die Zebrafisch, von Larven 3,5 mm Standard-Länge (SL) mit einem Herzen aus 100 um Ventrikels (VL), für Erwachsene, mit SL von 30mm und VL 1 mm oder größer. Larven und ausgewachsene Fische haben ganz unterschiedliche Körper-und Organ-Morphologie. Die Larven sind nicht nur deutlich kleiner, sie haben weniger Pigment und jedes Organ ist optisch sehr schwer zu identifizieren. Aus diesem Grund verwenden wir verschiedene Techniken Dissektion. Wir verwendeten Pre-Dissektion der Fixierung, als wir entdeckten, dass Herz direkt nach euthanization seziert eine variable Morphologie haben, mit sehr locker und Ballon wie Atrien im Vergleich mit Herzen entfernt folgende Fixierung. Der Fisch vor dem Tag Dissektion, behalten in vivo Morphologie und Kammer-Position (Daten nicht gezeigt). Darüber hinaus ist für Demonstrationszwecke, nutzen wir das Herz (Herzinfarkt) spezifische GFP transgenen Tg (myl7: GFP) twu34 (12), die uns das gesamte Herz visualisieren können und ist besonders nützlich in einem frühen Stadium in der Entwicklung, wenn das Herz Morphologie ist weniger deutlich vom umgebenden Gewebe. Präparation des Herzens macht eine weitere Analyse der Zell-und Molekularbiologie zugrunde liegenden Herz Entwicklung und Reifung mit in-situ-Hybridisierung, Immunhistochemie, RNA-Extraktion oder andere analytische Methoden leichter in post-embryonale Zebrafisch. Dieses Protokoll wird eine wertvolle Technik für die Untersuchung der kardialen Entwicklung Reifung und Alterung.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Eine klare, standardisierte Methode zur Präparation und Isolierung des Zebrafisch-Herzen an mehreren Entwicklungsstufen beschrieben sind. Annotation und Quantifizierung Techniken werden ebenfalls diskutiert.

Herz Dissection in Larven, juvenile und adulte Zebrafisch, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

21.3K Aufrufe.  Queens College, City University of New York. Eine klare, standardisierte Methode zur Präparation und Isolierung des Zebrafisch-Herzen an mehreren Entwicklungsstufen beschrieben sind. Annotation und Quantifizierung Techniken werden ebenfalls diskutiert.

Serie: Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissection der Larven ZNS von Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Forschung

Biologie

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila Larven NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Gross und Fine Dissection of Inner Ear Sinnesepithelien in Adult Zebrafisch ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissection von Organen aus dem Adult Zebrafisch

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

Dissection und Färbung von Drosophila Larven Ovarien

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Präparation des Adult Zebrafisch Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Regelmäßige Pflege und Wartung eines Zebrafisch (<em&gt; Danio rerio</em&gt;) Labor: Eine Einführung

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modellierung des Morbid Human Genome mit Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Adult Zebrafisch

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...