Name: whole mount dissection and immunofluorescence of the adult mouse cochlea
Uploaded: 2016-01-01T15:00:00
Description: Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

This work was supported in part by a grant from the Office of Naval Research (N000141310569). The Southern Illinois University School of Medicine Research Imaging Facility equipment was supported by the National Center for Research Resources-Health (S10RR027716).

Ethiek Verklaring: Procedure waarbij proefdieren zijn door de Institutional Animal Care en gebruik Comite aan de Southern Illinois University School of Medicine. 1. Winning van Temporal Bones <ol><li> Identificeren temporale botten in de onderkant van de muis schedel 13 en schraap de hersenzenuwen met behulp van standaard patroon tang. </li><li> Plaats standaard patroon tang op het puntje van de otic capsule en met de duim van de andere hand drukt op de achterste halfronde kanaa...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+plasmid+electroporation+of+the+murine+organ+of+Corti.">Primary culture and plasmid electroporation of the murine organ of Corti.</a> J Vis Exp. (36), e1685 (2010).</li><li>Burns, J. C., On, D., Baker, W., Collado, M. S., Corwin, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+half+the+hair+cells+in+the+mouse+utricle+first+appear+after+birth,+with+significant+numbers+originating+from+early+postnatal+mitotic+production+in+peripheral+and+striolar+growth+zones.">Over half the hair cells in the mouse utricle first appear after birth, with significant numbers originating from early postnatal mitotic production in peripheral and striolar growth zones.</a> J Assoc Res Otolaryngol. 13 (5), 609-627 (2012).</li><li>Collado, M. 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<html> Er zijn een aantal cruciale stappen voor een succesvolle ganse berg dissectie en immunokleuring. Echter voordat een van deze werkwijzen worden uitgevoerd, wordt goede fixatie van de cochleaire weefsel nodig. We raden het gebruik van methanol vrij, ultra-zuivere, EM leerjaar PFA. PFA uit poeder kan sporen van methanol en een instabiele pH die de kwaliteit van immunofluorescentie afneemt hebben. Andere groepen hebben ook aangetoond dat soortgelijke dissecties mogelijk met fixatieven die geen formaldehyde 14-16<...

De spiraalvormige slakkenhuis van het binnenoor, vervat in de temporale, herbergt het orgaan van Corti, het auditieve sensorische eindorgaan bij zoogdieren. De cochlea is tonotopically georganiseerd en gewoonlijk verdeeld in apicale, midden en basale bochten overeenkomen met verschillende frequentiegebieden met hoogfrequent geluid detectie in de basis en laagfrequente detectie in de apex 1. Haarcellen, de mechanosensorische cellen van het orgaan van Corti, lopen de lengte van het slakkenhuis, dat is ongeveer 6 mm lang in muizen 2,3. Deze cellen zet de mechanische energie van geluidsgolven, die worden uitgezonden via de met vloeistof gevulde membraneuze labyrint, in neurale signalen die worden verwerkt door de centrale auditieve structuren. De hier beschreven techniek verschaft een werkwijze voor het bereiden whole mounts van het orgaan van Corti na verkalking van het slakkenhuis is voltooid (voor monsters die variëren van één week tot volwassenheid). Wij stellen ook een werkwijze voor het immunostaining de hele gemonteerd cochleaire weefsel. Cochleaire whole mounts zijn cruciaal voor visualisatie van haarcellen en omliggende steuncellen in hun natuurlijke ruimtelijke indelingen en maken voor analyse in drie dimensies door het gebruik van confocale microscopie. Drs. Hans Engstrom en Harlow Ades oorspronkelijk beschreven geheel mount cochleair versnijdingsmethode in 1966. Zij gedetailleerd techniek snel oplossen en ontleden verkalkte cochleae ondergedompeld in vloeistof uit verschillende zoogdieren, behoud korte intacte segmenten van het orgaan van Corti voor microscopische analyse 4. De ontleding van een losgemaakte, verkalkte rat cochlea is ook geïllustreerd in een instructievideo 5. Drs. Barbara Bohne en Gary Harding aan de Washington University maakte een aantal belangrijke wijzigingen in deze methode. In hun versie van het cochleair hele berg methode, de temporale bot is ontkalkt, ingebed in kunststof, en vijf halve bochten of tien kwart-bochten waren dissected 6,7. Dr. Charles Liberman en collega&#39;s bij Eaton Peabody Laboratories, Massachusetts Eye en Ear Infirmary, bewerkt deze techniek, zodat plastic inbedding niet verplicht was 8. Verdere modificatie van de techniek zich heeft voorgedaan in het lab Dr Jian Zuo&#39;s in het St. Jude Children&#39;s Research Hospital 9-12 waarin de dissectie methode hier gepresenteerde geïnformeerd. We gebruiken een andere strategie om de toegang tot het orgaan van Corti dan Bohne en Liberman, die isolatie van volledige apicale, midden en basale bochten maakt krijgen. Zo is de ontleed weefsel is groter en minder waarschijnlijk worden verloren of beschadigd tijdens de dissectie of immunokleuring processen. Bovendien vergemakkelijkt de huidige methode van het meten van het apicale of basale uiteinde haak om een ​​frequentiegebied te identificeren. Hoewel veel labs voeren immunokleuring van cochleaire weefsel, het is onduidelijk waar deze methode is ontstaan. Dientengevolge zijn er diverse recepten voor het blokkeren buffers en antilichaam incubatie buffers die de prestaties van individuele primaire antilichamen kunnen beïnvloeden. Hier presenteren we een methode voor immunokleuring met fluorescent gelabelde antilichamen die toepassing meest gebruikte antilichamen in het auditieve veld. De complexe vorm van de cochlea, delicate structuur van het orgaan van Corti, en benige omhulsel vormen een uitdaging voor histologische en biochemische analyse. Verschillende technieken worden momenteel in het gebied gehoor deze moeilijke functies overwinnen en visualiseren van de cellen in het orgaan van Corti, elke techniek met zijn eigen voor- en nadelen. De hier gepresenteerde protocol maakt ganse berg dissectie van de volwassen muis cochlea en met kleine wijziging, potentieel kan worden gebruikt om de kritische structuren binnen het cochleae onderzoeken vanuit verschillende andere modelorganismen gebruikt in het veld.

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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 stellen een werkwijze voor het orgaan van Corti isoleren drie intacte cochleaire windingen (apex, midden en base) vanaf cochleaire weefsel dat verkalkt, met belangrijke dissectie stappen weergegeven in figuur 1. In de eerste postnatale week van ontwikkeling, verkalking van de muis cochlea onvolledig en een eenvoudigere versnijdingsmethode kan worden gebruikt 13. Met behulp van de neonatale hele berg versnijdingsmethode met slakkenhuis van P7 en ouder muizen...

Watch this Scientific Journal Video about Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea at JoVE.com

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

We presenteren een methode om de volwassen orgaan van Corti isoleren drie intacte cochleaire windingen (apex, midden en base). We tonen ook een procedure voor immunokleuring met fluorescent gelabelde antilichamen. Samen technieken maken het bestuderen van haarcellen, steuncellen en andere celtypen in de cochlea.

Hele Mount Dissection en Immunofluorescentie van de 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 ...

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A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

Een eenvoudige één stap Dissection protocol voor Whole-mount voorbereiding van Adult<em&gt; Drosophila</em&gt; Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...