Name: whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna
Uploaded: 2014-05-04T14:00:00
Description: Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

Denne studien ble støttet av mext-støttede Program for Strategic Research Foundation på private universiteter og JSPene Young Scientist B stipend for HT Vi ønsker å takke Ohtake Norihito å redigere videoklippene.

En. Forbered eple plate <ol><li> Bland 12,5 g agar, 125 ml 100% kommersielt tilgjengelig eplejuice, 12,5 g glukose og 375 ml H2O Micro blandingen i 1 til 2 minutter, og helles til 3 cm celledyrkningsskål. Oppbevar ved 4 ° C. </li></ol> 2. Genetisk kryss <ol><li> Bruk følgende representant genetisk kryss: </li></ol> Or49a-mCD8 :: GFP / CYO x w 1118 Tre. Dissection og farging protokollen <ol><li> Anesthetiz...

<ol>
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Den disseksjon av Drosophila antennen beskriver vi er enkel og lett å utføre i et laboratorium setting. For å sikre en vellykket disseksjon, er det viktig å utnytte fin-edged saks. Mens farging av den dissekerte antenne, er det viktig å inkubere dem i en fuktighetsfylt beholder for å unngå fordamping av antistoffløsningen. Den dissekert antennen har en tendens til å flyte i løsningen. Ved hjelp av 0,1% Triton i PBS under fiksering og blokkeringstrinn som vil lette nedsenking av antennen i løsningen, ...

Den olfaktoriske system blir brukt til å skille mellom et mangfold av luktmolekyler og deretter sende den resulterende informasjon til de høyere hjerne sentre. Denne inngangen brukes til nøyaktig kontroll grunnleggende dyreatferd, for eksempel fôring og parring 1-6. Som hver lukte nevron type er forbundet med et bestemt sett av lukt, spredning av olfactory reseptor nevroner (ORN) s er avgjørende for riktig luktsystemet funksjon 7. Drosophila genetikk gjør oss i stand til å utføre enkelt celle nivå etterforskningen involverer molekylære mekanismene forbundet med ORN utvikling og fysiologiske funksjon 8-16. Hele fjellet farging av Drosophila antenner har gjort oss i stand til å forstå i større detalj de molekylære mekanismene som er involvert i spredning av olfactory reseptor nevroner (ORN) s 7. Heri vi gi en omfattende beskrivelse av en enkel metode for å achieve dette.

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	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Stemi DV4 dissection microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:113px;">Stemi DV4</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Glass bottom culture dishes&#160;</td>
			<td style="width:143px;">MatTek corporation</td>
			<td style="width:113px;">P35G-0-10-C</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Dissection scissor</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:113px;">15000-08</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Rat anti-ELAV</td>
			<td style="width:143px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:113px;">7E8A10</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mouse anti-GFP</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:113px;">A11122</td>
			<td style="width:116px;">Dilution 1:400</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rabbit IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">711-225-152</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rat IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">712-165-150</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
	</tbody>
</table>

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

Odorant molekyler binder seg til sine mål reseptorer på en presis og koordinert måte. Hver reseptor gjenkjenner et bestemt signal, og videresender denne informasjonen til hjernen. Som sådan, å bestemme hvor lukte informasjon overføres til hjernen, å modifisere både persepsjon og oppførsel, fordeler undersøkelse. Interessant, det er nye bevis på at mobilnettet transduksjon og transkripsjonsfaktorer er involvert i spredning av olfactory reseptor nervecellen. Her gir vi en robust hel montere immunologisk merking metoden til analysen in vivo olfactory reseptor nervecellen organisasjon. Ved hjelp av denne metoden, identifiserte vi alle olfactory reseptor nerveceller med anti-ELAV antistoff, et kjent pan-nevrale markør og Or49a-mCD8 :: GFP, en olfactory reseptor nervecellen spesifikt uttrykt i NBA nevron bruker anti-GFP antistoff.

Sikre at både disseksjon og fiksering utføres raskt er en nøkkelfaktor for å oppnå suksess med denne protokollen. Ved hjelp av gode saks og pinsett er også avgjørende. Etter farging, ble fluorescerende merket antenner undersøkt under et konfokalmikroskop. Vi normalt ta 1/iM seksjoner ved hjelp av et 20x objektiv. Vi merket Nba 7 Orns bruker Or49a-mCD8 :: GFP og telte antall NBA Orns i vill-type antenne. Den mCD8-GFP reporter er cellemembranen lokalisert og slik uttrykket ses på figur 2 utstillinger c...

Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna (Video) | JoVE

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.

Hele Mount immunolabeling av olfactory Receptor nevroner i<em&gt; Drosophila</em&gt; Antenne

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

Research

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Neuroscience

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

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Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

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DII-Merking av DRG Neurons til Study aksonal Avgrening i en hel Mount Utarbeidelse av Mouse Embryonic Spinal Cord

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

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Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

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Appetitive Assosiative Olfactory læring i<em&gt; Drosophila</em&gt; Larver

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

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Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

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The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

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Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

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Hele Mount Dissection og Immunfluorescens av 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 ...