Gli autori desiderano ringraziare il Dr. Louis Reichardt per i topi TrkA taulacZ e Dr. Annette Markus per la discussione penetranti e suggerimenti. Questo lavoro è stato sostenuto da fondi di avvio dalla Fondazione Burke e assegno di ricerca della Fondazione Whitehall 2010-08-61, un assegno di ricerca da Wings for Life Foundation (WFL-US-028/14), concedere ZB1-1102-1 dal Christopher e Dana Reeve Foundation, e le sovvenzioni 1R01EY022409 e 3R01EY022409-01S1 dal National Eye Institute, a JZ. KJO è un collega Goldsmith.

NOTA: Tutte le procedure sono conformi alla Guida NIH per l&#39;uso e la cura di animali da laboratorio. Il protocollo animale è stato approvato dal IACUC al Weill Cornell Medical College. 1. Preparazione del tessuto <ol><li> Euthanize femmine scaduta a gravidanza per dislocazione cervicale 15. Sezionare embrionali E16 - E18 embrioni da femmine scaduta a gravidanza e posto embrioni singolarmente nei pozzetti di un piatto 6-bene, pieni di fosfato salino freddo tamponata (PBS). </li><li> Sciacquare embrioni in PBS freddo. </li><li> Rimuovere una piccola porzione della membrana amniotica dell&#39;embrione e posto in una soluzione di proteinasi K pre-preparato per la successiva genotipizzazione. In alternativa, se la membrana amniotica è perso, rimuovere una piccola porzione della punta della coda dell&#39;embrione e metterlo in soluzione proteinasi K </li><li> Poke i fori nella pelle in tutto l&#39;embrione utilizzando perni di insetti (almeno 100 per lato). </li><li> Rimuovere PBS e aggiungere lentamente fresco 2% paraformaldeide (PFA),pH 7.4, al pozzo. </li><li> Agitare delicatamente per 2 ore a 4 ° C. </li><li> Risciacquare embrioni due volte in PBS e conservare in PBS a 4 ° C fino colorazione. </li></ol> 2. Genotipizzazione <ol><li> Immergere membrane amniotiche o croce embrione in proteinasi K miscela secondo le istruzioni del produttore. </li><li> Incubare a 56 ° C per 10 min. </li><li> Estrarre DNA utilizzando un kit di purificazione di DNA disponibile in commercio. </li><li> Prendere 0,5 l di totali 200 microlitri purificati soluzione di DNA genomico, combinarlo con 0,5 ml di ciascuna delle senso e antisenso primer specifici geni (10 micron), 2 microlitri dNTP Mix (2,5 mm di dATP, dCTP, dGTP e dTTP) , 0,2 microlitri polimerasi Taq (5 U / ml) e 2 microlitri tampone PCR (10x), aggiungere H 2 O per un volume totale di 20 microlitri. NOTA: sequenze primer sono le seguenti (Tabella 1): TrkA: TrkA_F: GCG GGC GCC GCC GCG ATG, TrkA_R: GAA GCC GCC TGC GCG GCT CTG CCA GGG TG; PCR fragLunghezza mento: 400 paia di basi (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 lunghezze frammento: wild type, 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 frammento lunghezza: 360 bp. </li><li> Eseguire un PCR con il seguente programma per TrkA: passo 1: 1 min a 95 ° C, punto 2: 10 sec a 95 ° C, punto 3: 20 sec a 68 ° C, il punto 4: 30 sec a 72 ° C. Ripetere i punti 2 - 4 per 40 cicli. <ol><li> Per LacZ e Bax, eseguire una PCR con il seguente programma: punto 1: 1 min a 95 ° C, punto 2: 10 sec a 95 ° C, punto 3: 1 min a 54 ° C, il punto 4: 1 min a 72 ° C, ripetere i punti 2 - 4 per 35 cicli. </li></ol></li><li> Campioni Run PCR su un gel di agarosio 2% a 100 V in 1x tampone Tris-acetato-EDTA per 20 minuti con una corsia di scala DNA per valutare lunghezze frammento. </li><li> Analizzare gli embrioni per la presenza di tipo selvaggio TrkA, TRKA-tauLacZ e Bax alleli nulli. I genotipi di embrioni sperimentali sono TrkA taulacZ / WT: Bax - / - e TrkA taulacZ / taulacZ: Bax - / -, a seconda delle questioni sperimentali. Gli embrioni privi del giornalista tauLacZ saranno negativi per assonale colorazione e può quindi servire come controlli negativi per calibrare il processo di colorazione. </li></ol> 3. Embryo Colorazione <ol><li> Utilizzare un kit commerciale per lacZ colorazione con alcune modifiche come descritto di seguito nelle sezioni 3,2-3,8. Qui, utilizzare il kit Millipore. </li><li> Immergere embrioni in tampone A per almeno 1 ora, scuotendo leggermente a temperatura ambiente. </li><li> Direttamente immergere embrioni in tampone B per almeno 15 minuti, agitando dolcemente alla temperatura ambiente. </li><li> Mentre gli embrioni sono in Buffer A / B, soluzione Tissue Base pre-caldo a 37 ° C. Utilizzare 5 ml di soluzione di tessuto di base per embrione. </li><li> Mentre gli embrioni sono in Buffer A / B, preparare fresco 5-bromo- 4- cloro- 3- indolil α-D-galattopiranoside (X-gal) ad una concentrazione di 40 mg / ml in 100% dimetilsolfossido (DMSO). </li><li> Diluire X-gal nella soluzione Tissue Base pre-riscaldato ad una concentrazione di 1 mg / ml e si aggiungono 5 ml di X-gal / miscela Base Tissue in una fiala di scintillazione in vetro. </li><li> Trasferire gli embrioni in una fiala di scintillazione di vetro contenente miscela Base X-gal / tessuto. Sigillare coperchio con Parafilm e incubare a 37 ° C durante la notte, controllando la presenza di blu macchia. </li><li> Embrioni Postfix con freschi 4% PFA, pH 7.4. Agitare delicatamente per 4 ore a 4 ° C. </li><li> Sciacquare embrioni due volte in PBS freddo. </li></ol> 4. Tissue Disidratazione e Clearing <ol><li> Luogo embrioni in metanolo al 50% al 50% della miscela / PBS per 30 minuti a temperatura ambiente, agitando in fiale di vetro. </li><li> Continuare a disidratare in 75% metanolo / 25% PBS per 30 minuti a temperatura ambiente agitando in flaconcini di vetro. </li><li> Continuare a disidratare in metanolo al 90% / 10% PBS per 30 min a temperatura ambiente agitando in flaconcini di vetro. </li><li> Continuare a disidratare in metanolo 100% per 30 min (2x) a temperatura ambiente agitando in flaconcini di vetro. </li><li> Mentre embrioni sono disidratazione, preparare un 1: 1 (v: v) miscela di alcool benzoile e benzoil benzoato. NOTA: alcol benzoile e benzoil benzoato è tossico. </li><li> Immergere embrioni in 1: 1 (v: v) miscela di metanolo 100%: 100% BABB per 15 min. Utilizzare il vetro solo perché il BABB si dissolverà in plastica. </li><li> Immergere embrioni in 100% BABB completamente chiaro il tessuto e visualizzare X-gal colorazione nell&#39;embrione eliminato. NOTA: L&#39;immagine appena possibile perché la macchia X-gal sbiadiscono nel tempo. </li></ol> 5. intero monte Imaging <ol><li> Stabilizzare l&#39;embrione, immerso in 100% BABB, agli angoli appropriati per fotografia con pinze di metallo. Illuminare utilizzando una combinazione di up-light e sotto-light fonti. </li><li> Acquisire le immagini con un microscopio dissezionedotato di una fotocamera digitale. Prendere esposizioni multiple in campo chiaro a cinque piani focali successivi 0,5 millimetri di distanza lungo l&#39;asse Z. I tempi di esposizione e f-stop possono variare a seconda di illuminazione e telecamere specifiche e devono essere ottimizzati per ogni installazione. </li><li> Utilizzare software standard di elaborazione delle immagini per elaborare immagini sovrapposte e generare fotomontaggi. </li></ol>

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The authors declare that there are no competing interests.

La procedura di colorazione X-gal sopra descritto di topi embrionali TrkA taulacZ permette la visualizzazione rapida e dettagliata delle proiezioni assoni lunga distanza nell&#39;embrione fisso intatto. A causa del fondo nulla Bax questi topi permettono di sondare di meccanismi di segnalazione che possono contribuire sia alla crescita degli assoni e la sopravvivenza neuronale. Accoppiamento con transgenici o knockout mice di interesse consente per la valutazione complessiva di fenotipi asson...

Creazione di precise reti neuronali è un complesso processo evolutivo essenziale per la funzionalità del sistema nervoso. Disturbi in questo processo porta a una disfunzione neuronale che è stato coinvolto in malattie neurologiche umane 1-3. Per studiare i meccanismi molecolari alla base della crescita degli assoni e di destinazione innervazione nei mammiferi, abbiamo sviluppato un protocollo per visualizzare le traiettorie assonali di TrkA-esprimere i neuroni sensoriali utilizzando una combinazione di due linee di topi geneticamente modificati. TrkA è un recettore per il fattore di crescita nervoso NGF ed è un marcatore funzionale dei neuroni sensoriali nocicettivi 4. TrkA è altamente espresso nei neuroni nocicettivi durante lo sviluppo precoce e media di sopravvivenza dei neuroni NGF-dipendente, la crescita degli assoni, arborization e destinazione innervazione 5-9. Nei topi TrkA taulacZ, il tipo selvatico gene TrkA è sostituito da un&#39;espressione taulacZ cassette 10, tale che la morfologia assonale dei neuroni TrkA-positive putativi può essere visualizzata dal β-gal (X-gal) colorazione 11. Usando una linea TrkA taulacZ / WT eterozigote, possiamo esaminare i fattori che possono regolare o interferire con lo sviluppo di sensoriali proiezioni afferenti in vivo. Inoltre, l&#39;espressione TrkA è assente in omozigoti topi TrkA taulacZ / taulacZ, che possono quindi essere utilizzati per valutare la crescita degli assoni promuovere meccanismi in assenza di segnalazione NGF / TrkA. Poiché i neuroni nocicettivi dipendono NGF / TrkA segnalazione non solo per la crescita degli assoni, ma anche per la sopravvivenza, ci avvaliamo di un&#39;altra linea di topi, manca la pro-apoptotico gene Bax, di inibire l&#39;apoptosi nei neuroni DRG embrionali, sottraendoli morte cellulare che è altrimenti osservata in assenza di segnalazione TrkA. La Bax - / - sfondo 12 consente quindi per la molecudissezione lar di vie di segnalazione che riguardano specificamente assone 7-9,13-15 crescita. In TrkA - / -: Bax - / - mice, neuroni DRG sopravvivono, ma sensoriale innervazione afferente nella pelle è completamente aboliti 14,15. Siamo in grado di attivare selettivamente vie di segnalazione per determinare i rispettivi contributi allo sviluppo delle proiezioni assoni. L&#39;utilità di questo metodo è che permette la valutazione delle variazioni di fenotipi di crescita assonale quando diverse modificazioni genetiche sono allevati sul TrkA taulacZ / taulacZ: Bax - / - o TrkA taulacZ / WT: Bax - / - sfondi.

<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

La visualizzazione del full-length proiezioni neuronali in embrioni è essenziale per acquisire una comprensione di come mammiferi reti neuronali si sviluppano. Qui si descrive un metodo per etichettare in situ un sottoinsieme di gangli delle radici dorsali (DRG) proiezioni assoni di valutare le loro caratteristiche fenotipiche con diverse linee di topi geneticamente manipolati. I neuroni TrkA-positivi sono neuroni nocicettori, dedicati alla trasmissione di segnali di dolore. Utilizziamo una linea di topi TrkA taulacZ etichettare le traiettorie di tutti assoni periferici TrkA-positivo nel topo embrione intatto. Abbiamo inoltre alleviamo la linea TrkA taulacZ su uno sfondo nullo Bax, che abolisce in sostanza l&#39;apoptosi neuronale, al fine di valutare le domande di crescita relative indipendentemente possibili effetti delle manipolazioni genetiche sulla sopravvivenza neuronale. Successivamente, i topi geneticamente modificati di interesse sono allevati con il TrkA TaulLinea nullo ACZ / Bax e sono quindi pronti per lo studio utilizzando le tecniche qui descritte. Questa presentazione include informazioni dettagliate sui piani di allevamento del mouse, la genotipizzazione al momento della dissezione, preparazione del tessuto, colorazione e di compensazione per consentire la visualizzazione del full-length traiettorie assonali in tutto il montaggio di preparazione.

I genotipi di TrkA WT / taulacZ: Bax - / - e TrkA taulacZ / taulacZ: Bax - / - gli embrioni possono essere inequivocabilmente determinati dal genotipo normale PCR (Figura 1). Display colorazione X-gal dettagliate pergole assonale periferici per via sottocutanea in embrioni convenzionalmente macchiati (figure 2, 3 bis), e in tutto l&#39;embrione dopo schiarimento del tessuto (figure 3b, 4). Abbiamo ...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

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 di embrione di topo Sensory Axon Proiezioni

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 Views.  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.

Video: 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-topo Imaging neuroanatomici Risonanza Magnetica

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

Ricerca

Neuroscienze

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.

Dissezione di un occhio mouse per un monte intero di dell&#39;epitelio pigmentato retinico

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.

Riproducibile nervo sciatico Crush mouse e successiva valutazione da parte della Rigenerazione intera analisi Muscle Monte

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.

Tutta Monte immunofluorescenza del mouse Retina neonatale per Indagare Angiogenesi<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.

Tutta Monte immunomarcatura di recettore olfattivo neuroni nel<em&gt; Drosophila</em&gt; Antenna

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.

Appartamento Monte Imaging di pelle di topo e la sua applicazione alla analisi del follicolo pilifero patterning e sensoriale Axon Morfologia

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; Dinamica a cono fotorecettore Axon Terminali della retina di topo

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.

Tutta Monte dissezione e immunofluorescenza del topo adulto Coclea

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 ...

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.

Tomografia a coerenza ottica: Imaging Mouse ganglionari cellule In Vivo

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

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Quadrupla Immunostaining della lampadina olfattiva per la visualizzazione di codici di identificazione molecolare Axon sensoriale olfattiva

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...