Die Autoren danken Dr. Louis Reichardt für die TrkA taulacZ Mäusen und Dr. Annette Markus für aufschlussreiche Diskussionen und Anregungen. Diese Arbeit wurde durch Mittel aus dem Startup-Burke-Stiftung sowie Whitehall-Stiftung Forschungsstipendium 2010-08-61, ein Forschungsstipendium von Wings for Life Foundation (WFL-US-028/14) unterstützt wird, gewähren ZB1-1102-1 von der Christopher &amp; Dana Reeve Foundation, und Zuschüsse 1R01EY022409 und 3R01EY022409-01S1 vom National Eye Institute, zu JZ. KJO ist ein Goldsmith Kerl.

Hinweis: Alle Verfahren mit dem NIH Leitfaden für die Benutzung und Pflege von Labortieren entsprechen. Das Tier Protokoll wurde von der IACUC am Weill Cornell Medical College zugelassen. 1. Gewebepräparation <ol><li> Euthanize timed-Schwangerschaft Frauen durch Genickbruch 15. Präparieren embryonalen E16 - E18 Embryonen aus timed-Schwangerschaft Weibchen und Ort Embryonen einzeln in die Wells einer 6-Well-Platte, mit kalter phosphatgepufferter Salzlösung (PBS) gefüllt. </li><li> Spülen Embryonen in kaltem PBS. </li><li> Entfernen eines kleinen Teils der Amnionmembran des Embryos und in eine vorbereitete Proteinase K-Lösung für die nachfolgende Genotypisierung. Alternativ, wenn die Amnionmembran verloren geht, nehmen Sie einen kleinen Teil der Spitze des Embryos Schwanz und ihn in Proteinase K-Lösung </li><li> Poke Löcher in die Haut rund um den Embryo mit Insektenstifte (mindestens 100 pro Seite). </li><li> Entfernen PBS und langsam frisch 2% Paraformaldehyd (PFA),pH 7,4, zum Brunnen. </li><li> Schütteln für 2 h bei 4 ° C. </li><li> Embryonen zweimal Spülen in PBS und lagern in PBS bei 4 ° C bis zur Färbung. </li></ol> 2. Genotypisierung <ol><li> Tauchen Amnionmembranen oder Embryo Schwänze in Proteinase K Mischung gemäß den Anweisungen des Herstellers. </li><li> Inkubiere bei 56 ° C für 10 min. </li><li> Extrahieren DNA unter Verwendung eines kommerziell erhältlichen DNA-Reinigungskits. </li><li> Nehmen 0,5 ul der gesamten 200 ul gereinigte genomische DNA-Lösung, kombiniert mit 0,5 &amp; mgr; l von jeder der genspezifischen Sense- und Antisense-Primer (10 &amp; mgr; M), 2 &amp; mgr; l dNTP-Mix (2,5 mM dATP, dCTP, dGTP und dTTP) , 0,2 ul Taq-Polymerase (5 U / ul) und 2 ul PCR-Puffer (10x), fügen H 2 O auf ein Gesamtvolumen von 20 ul. HINWEIS: Die Primersequenzen sind wie folgt (Tabelle 1): TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR fragment Länge: 400 Basenpaare (bp). Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, NeoR: CCG CTT CCA TTG CTC AGC GG; PCR-Fragment Längen: Wildtyp, 304 bp; Bax null, 507 bp. TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR-Fragment-Längen: 360 bp. </li><li> Führen einer PCR mit dem folgenden Programm für TrkA: Schritt 1: 1 min bei 95 ° C, Stufe 2: 10 sec bei 95 ° C, Stufe 3: 20 sec bei 68 ° C, Stufe 4: 30 sec bei 72 ° C. Wiederholen Sie die Schritte 2-4 für 40 Zyklen. <ol><li> Für LacZ und Bax, führen eine PCR nach folgendem Programm: Schritt 1: 1 min bei 95 ° C, Stufe 2: 10 sec bei 95 ° C, Stufe 3: 1 min bei 54 ° C, Schritt 4: 1 min bei 72 ° C, wiederholen Sie die Schritte 2-4 für 35 Zyklen. </li></ol></li><li> Run PCR-Proben auf einem 2% Agarosegel bei 100 V in 1x Tris-Acetat-EDTA-Puffer für 20 min mit einer Spur-DNA-Leiter, um Fragmentlängen abzuschätzen. </li><li> Analysieren Embryonen für die Anwesenheit von Wildtyp-TrkA, TRKA-tauLacZ und Bax Nullallele. Die Genotypen der experimentellen Embryonen TrkA taulacZ / WT: Bax - / - und TrkA taulacZ / taulacZ: Bax - / - in Abhängigkeit von den experimentellen Fragen. Embryonen ohne die tauLacZ Reporter negativ für axonalen Färbung sein und kann daher dazu dienen als Negativkontrollen, um den Färbeprozess zu kalibrieren. </li></ol> 3. Embryo Färbung <ol><li> Verwenden eines kommerziellen Kits zur LacZ-Färbung mit einigen Änderungen, wie unten in den Abschnitten 3.2 - 3.8. Hier verwenden Sie die Millipore-Kit. </li><li> Tauchen Embryonen in Puffer A für mindestens 1 h bei Raumtemperatur vorsichtig schütteln. </li><li> Direkt tauchen Embryonen in Puffer B für mindestens 15 min bei Raumtemperatur vorsichtig schütteln. </li><li> Während Embryonen werden in Puffer A / B, vorwärmen Tissue Basislösung bei 37 ° C. Verwenden Sie 5 ml Gewebebasislösung pro Embryo. </li><li> Während Embryonen in Puffer A / B, bereiten Sie frisches 5-bromo- 4- Chlor- 3- indolyl α-D-galactopyranosid (X-gal) bei einer Konzentration von 40 mg / ml in 100% Dimethylsulfoxid (DMSO). </li><li> Verdünne X-gal in die vorgewärmte Tissue Basenlösung mit einer Konzentration von 1 mg / ml und 5 ml X-gal / Tissue Basismischung auf eine Glasszintillationsröhrchen. </li><li> Übertragen Embryonen auf ein Glasszintillationsröhrchen enthaltende X-gal / Tissue Basismischung. Seal Deckel mit Parafilm und bei 37 ° C über Nacht, Prüfen auf Vorhandensein von Bläue. </li><li> Postfix Embryonen mit frischen 4% PFA, pH-Wert 7,4. Schütteln 4 Stunden bei 4 ° C. </li><li> Embryonen zweimal Spülen in kaltem PBS. </li></ol> 4. Tissue Dehydration und Clearing <ol><li> Ort Embryonen in 50% Methanol / 50% PBS Gemisch 30 min bei Raumtemperatur in den Glasphiolen Schütteln. </li><li> Weiterhin in 75% Methanol / 25% PBS für 30 Minuten bei Raumtemperatur dehydriert, in Glasfläschchen schüttelnd. </li><li> Weiterhin in 90% entwässern Methanol / 10% PBS 30 Minuten lang bei Raumtemperatur in Glasfläschchen schüttelnd. </li><li> Weiter in 100% Methanol für 30 min (2x) bei Raumtemperatur zu entwässern, in Glasfläschchen schüttelnd. </li><li> Während Embryonen Dehydratisierung, bereiten ein 1: 1 (v: v) Mischung aus Benzoylperoxid Alkohol und Benzoylbenzoat. HINWEIS: Benzoyl Alkohol und Benzoylbenzoat ist giftig. </li><li> Tauchen Embryonen in 1: 1 (v: v) Mischung aus 100% Methanol: 100% BABB für 15 min. Benutzen Glas nur, weil die BABB wird Kunststoff auflösen. </li><li> Tauchen Embryonen in 100% BABB völlig klar, dass die Gewebe und X-Gal-Färbung in der gelöscht Embryo sichtbar zu machen. HINWEIS: Bild so schnell wie möglich, weil die X-gal-Färbung mit der Zeit verblassen. </li></ol> 5. Ganze Berge Imaging <ol><li> Stabilisierung des Embryos, in 100% BABB getaucht, an den entsprechenden Winkel für die Fotografie mit Metallzangen. Beleuchten Sie mit einer Kombination aus bis-Licht und unter Lichtlichtquellen. </li><li> Erwerben Sie Bilder mit einem Binokularmit einer Digitalkamera ausgestattet. Nehmen Sie mehrere Hellfeldaufnahmen an fünf aufeinander folgenden Brennebenen 0,5 mm, die entlang der Z-Achse. Belichtungszeiten und Blendenstufen können nach Beleuchtung und Kameradaten variieren und müssen für jedes Setup optimiert werden. </li><li> Verwenden Sie Standard-Bildverarbeitungs-Software, um Overlay-Bilder verarbeiten und Fotomontagen. </li></ol>

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The authors declare that there are no competing interests.

Die oben beschriebene X-gal Färbeverfahren embryonaler TrkA taulacZ Mäusen erlaubt die schnelle und präzise Visualisierung Fern Axon in der intakten Fest Embryo. Aufgrund der Bax null Hintergrund diese Mäuse ermöglichen die Untersuchung von Signalmechanismen, die sowohl Axon Wachstum und Überleben von Neuronen beitragen können. Paarung mit transgenen oder Knockout-Mäuse von Interesse ermöglicht die umfassende Beurteilung der axonalen Phänotypen und kann als nützlicher Leitfaden f?...

Gründung der genauen neuronalen Netzen ist eine komplexe Entwicklungsprozess wesentlich für die Funktion des Nervensystems. Störung in diesem Verfahren führt zu neuronalen Funktionsstörungen, die in menschlichen neurologischen Erkrankungen 1-3 gebracht wurde. Um die zugrunde liegenden molekularen Mechanismen der Axon Wachstum und Ziel Innervation bei Säugetieren zu untersuchen, haben wir ein Protokoll, um die axonalen Bahnen der TrkA-exprimierenden sensorischen Neuronen mit einer Kombination von zwei genetisch veränderten Mauslinien zu visualisieren entwickelt. TrkA ist ein Rezeptor für den Nervenwachstumsfaktor NGF und ein Funktionsmarker nozizeptiven sensorischen Neuronen 4. TrkA ist stark in nozizeptiven Neuronen während der frühen Entwicklung ausgedrückt und vermittelt NGF-abhängige Überleben von Neuronen, das Wachstum von Axonen, Verzweigung und Ziel Innervation 5-9. In TrkA taulacZ Mäusen wird die Wildtyp-TrkA-Gen von einem taulacZ Ausdruck c ersetztassette 10, derart, dass die axonale Morphologie putative TrkA-positive Neurone kann durch β-Gal (X-gal) Anfärben 11 visualisiert werden. Mit einem heterozygot TrkA taulacZ / WZ-Linie, können wir Faktoren, die zu regulieren oder zu stören die Entwicklung der sensorischen afferenten Projektionen in vivo kann zu untersuchen. Außerdem ist TrkA Expression in homozygoten TrkA taulacZ / taulacZ Mäusen, die daher verwendet werden können, um das Wachstum der Axone zu fördern Mechanismen in Abwesenheit von NGF / TrkA Signalisierungs beurteilen abwesend. Seit nozizeptiven Neuronen hängt von NGF / TrkA Signalisierung nicht nur für das Wachstum von Axonen, sondern auch für das Überleben, setzen wir einen weiteren Mauslinie, ohne die pro-apoptotische Bax-Gens, die Apoptose in embryonalen DRG-Neuronen hemmen, retten sie vor Zelltod, die sonst ist in Abwesenheit von TrkA Signalisierungs beobachtet. Das Bax - / - Hintergrund 12 ermöglicht somit die Molekular Dissektion von Signalwegen, die speziell beeinflussen Axonwachstum 7-9,13-15. In TrkA - / -: Bax - / - Mäuse, DRG-Neuronen überleben, aber sensorischen afferenten Innervation der Haut vollständig abgeschafft 14,15. Wir können selektiv aktiviert Signalwege auf ihre jeweiligen Beiträge zur Entwicklung von Axon Projektionen zu bestimmen. Der Nutzen dieser Methode ist, dass sie die Beurteilung der Änderungen im Wachstum der Nervenfasern Phänotypen, wenn verschiedene genetische Veränderungen werden auf die TrkA taulacZ / taulacZ gezüchtet: Bax - / - oder TrkA taulacZ / WT: Bax - / - Hintergründe.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

Die Visualisierung der in voller Länge neuronalen Projektionen in Embryonen ist wesentlich für ein Verständnis, wie Säugetier neuronale Netze entwickeln zu gewinnen. Hier beschreiben wir eine Methode, um in situ zu markieren eine Teilmenge der Hinterwurzelganglien (DRG) Axon ihrer phänotypischen Eigenschaften beurteilen mit mehreren genetisch manipulierte Mauslinien. Die TrkA-positive Neurone nociceptor Neuronen, für die Übertragung von Schmerzsignalen gewidmet. Wir nutzen eine TrkA taulacZ Mauslinie, die Flugbahnen aller TrkA-positiven peripheren Axonen im intakten Mausembryo zu kennzeichnen. Wir züchten ferner den TrkA taulacZ Schnur auf eine Bax null Hintergrund, die im Wesentlichen aufhebt neuronale Apoptose, um wachstumsbedingte stellen unabhängig von möglichen Wirkungen von genetischen Manipulationen auf das neuronale Überleben zu beurteilen. Anschließend werden gentechnisch veränderte Mäuse von Interesse mit der TrkA Taul gezüchtetlacZ / Bax Nulllinie und sind dann bereit für die Studie unter Verwendung der hierin beschriebenen Techniken. Diese Präsentation enthält detaillierte Informationen über Maus Zuchtpläne, Genotypisierung bei der Dissektion, Gewebevorbereitung, Färbung und Clearing zur Visualisierung von voller Länge axonalen Bahnen ganz-mount Vorbereitung zu ermöglichen.

Die Genotypen von TrkA WT / taulacZ: Bax - / - und TrkA taulacZ / taulacZ: Bax - / - Embryonen können eindeutig durch Standard-PCR Genotypisierung bestimmt werden (Abbildung 1). X-Gal-Färbung zeigt detaillierte periphere axonale Verzweigungen subkutan in konventionell gefärbten Embryonen (Figuren 2, 3a), und während der Embryo nach Gewebe Clearing (3b, 4). / - - Linie mit Mäusen, di...

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Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Whole-Mount-Imaging von Mouse Embryo Sinnes Axon Projektionen

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K Aufrufe.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Serie: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Maus Neuroanatomische Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Forschung

Neurowissenschaften

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Präparation einer Maus Eye für einen ganzen Berg von retinalen Pigmentepithel

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Reproduzierbare Maus Ischiasnerv Crush und anschließende Bewertung der Regeneration von Whole mount Muskelanalyse

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Ganze Berge Immunfluoreszenzfärbung der Neonatal Maus Retina zu untersuchen Angiogenese<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Ganze Berge Immunmarkierung der olfaktorischen Rezeptorneuronen in der<em&gt; Drosophila</em&gt; Antennen

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Wohnung Berg-Imaging von Mäusehaut und ihre Anwendung auf die Analyse von Mustern und die Haarfollikel Sinnes Axon Morphologie

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imaging Ca<sup&gt; 2+</sup&gt; Dynamik in Cone Photorezeptor Axonterminalen des Maus-Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Ganze Berge Dissection und Immunfluoreszenz der erwachsenen Maus Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Dorsalwurzelganglion Injection und dorsale Wurzel Quetschverletzungen als Modell für Sensory Axonregeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optische Kohärenztomographie: Maus retinalen Ganglienzellen Zellen In Vivo Imaging

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...