Name: removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells
Uploaded: 2016-11-02T13:00:00
Description: Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

Vi takker Gary Laevsky for nyttige diskussioner om mikroskopi. Dette arbejde blev finansieret af NIH tilskud R01GM061107 og R01GM067758 til ERG

Bemærk: Proceduren for muskel fjernelse (figur 1) er en modifikation af tidligere beskrevne metoder til fremstilling af larver fileter. De skridt, før og efter fjernelse muskel er skitseret kort og læseren henvises til tidligere arbejde 10, 11 for mere detaljerede beskrivelser. 1. Dissekere Larve i Cold Saline <ol><li> Forbered en arbejdsgruppe fortynding af kold HL3.1 saltvand 15 eller koldt Ca 2+ -fri HL3.1 saltvand 11 <stro...

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Her en protokol er beskrevet for manuel fjernelse af muskelvæv fra Drosophila larver fileter. Denne protokol ændrer tidligere beskrevet larver dissektion teknikker 10,11. Efter larve dissekeres i en silikoneelastomer fad, er den dorsale midtlinje placeret. En enkelt pincet krog i sin fladeste mulig orientering, er omhyggeligt indsat mellem muskelvæv og epidermis, nær den dorsale midtlinje. Tangen er forsigtigt trækkes opad til separat muskelvæv fra et forankringspunkt i hvert larve segment af i...

Drosophila larval dendritiske arborization (DA) neuroner udgør en værdifuld model til undersøgelse neuronal udvikling på grund af deres modtagelighed for genetisk manipulation og den lethed, hvormed de kan afbildes. Disse sensoriske neuroner har været medvirkende til at identificere mange veje, der styrer dendritceller morfogenese 1-3. Fire klasser af DA-neuroner (klasse I - IV) innerverer larvestadium epidermis. Disse neuroner ligger mellem basalmembranen og epidermis, med deres dendritter danner stort set todimensionale arrays 4,5. Af de fire klasser, klasse IV da neuroner har de højest forgrenede dorne og ligesom sensoriske neuroner af andre dyr, udarbejdelsen af disse dorne kræver iboende faktorer samt stikord fra omkringliggende væv, især epidermis, for deres udvikling 6-9 . Undersøgelser til at bestemme, hvordan en sådan neuronal og ekstra neuronal kendsgerningors styre dendritceller morfogenese fordel af evnen til at detektere proteinekspression in situ ved immunofluorescens. Den ydre neglebånd af larve er uigennemtrængelig for antistoffer, men denne hindring er let overvindes ved udarbejdelsen af larver fileter gennem veletablerede dissektion metoder 10,11. Imidlertid kropsvæggen muskelvæv, der ligger lige indre til basalmembranen giver adskillige udfordringer hen imod visualisering af DA-neuroner og epidermisceller. Først, muskelvæv, hvilke linjer fleste af kroppens væg, i høj grad tilslører fluorescerende signaler stammer fra neuronal eller epidermalt væv. Dette reducerer væsentligt signal-støj-forhold i prøven. For det andet kan mange relevante proteiner udtrykkes i muskelvævet samt i neuroner eller epidermis. Denne muskel-afledte fluorescenssignal vil sandsynligvis yderligere obskure detektion af et fluorescenssignal fra neuron eller epidermis. Endelig fremskridt i mikroskopi teknologier ikkew tilladelse billeddannelse af prøver ved sub-diffraktion opløsning og kan være særligt nyttigt i kræsne lokalisering af proteiner, der er udtrykt i neuroner og omkringliggende epidermale celler 12,13. Men billedbehandling via super opløsning mikroskopi fordele fra et stærkt signal til støjforhold og nærhed af prøven til dækglasset. Ud over at reducere signalet til støjforholdet, larve organ væg muscle afstande DA-neuroner fra dækglasset og dermed begrænse den forbedrede billedopløsning, der kan opnås med super opløsning mikroskopimetoder. Udover udfordringer for immunfluorescens analyse, muskelvæv præsenterer en barriere for elektrofysiologiske optagelse fra sensoriske neuroner i larvestadiet kroppen væggen. Dens fjernelse derfor gavner neurofysiologisk manipulation af sensoriske neuroner 14. Her beskrives en fremgangsmåde til manuel fjernelse af Drosophila larvestadiet muskelvæv. Vi viser, at vores protokol permits immunfluorescens billeddannelse af proteiner, som ellers skjules af muskelvæv, forbedrer signal-støj-forhold for visualisering af klasse IV DA-neuroner, og muliggør anvendelse af super-opløsning mikroskopi til bedre skelne rumlige forhold af proteiner og cellulære strukturer i DA neuroner og epidermis.

<table border="0" cellpadding="0" cellspacing="0" width="1299">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dumont #5 tweezers</td>
			<td style="width:304px;">Electron Microscopy Sciences</td>
			<td style="width:151px;">72701-D</td>
			<td style="width:432px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Scissors, 8 cm, straight, 5 mm blades, 0.1 mm tips</td>
			<td>World Precision Instruments</td>
			<td>14003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 silicone elastomer kit</td>
			<td>Dow Corning</td>
			<td>3097358-1004</td>
			<td>for dissecting plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Austerlitz insect pins, 0.1 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fostec 8375 light source</td>
			<td>Artisan Technology Group</td>
			<td>62792-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Stemi 2000</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield antifade mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>for confocal microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prolong Diamond antifade mountant</td>
			<td>Life Technologies</td>
			<td>P36970</td>
			<td>for structured illumination microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro cover glass, 22x22 mm, No. 1.5</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superfrost Plus microscope slides, 25 x 75 x 1.0 mm</td>
			<td>Fisherbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Coracle antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>C615.16</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Discs large antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3</td>
			<td>supernatant, dilute 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit anti-dsRed antibody</td>
			<td>Clontech</td>
			<td>632496</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-rabbit antibody, Alexa Fluor 568 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11011</td>
			<td>dilute 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat anti-mouse antibody, Alexa Fluor 488 conjugated</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11001</td>
			<td>dilute 1:500</td>
		</tr>
	</tbody>
</table>

removal of drosophila muscle tissue from larval fillets for immunofluorescence analysis of sensory neurons and epidermal cells

Drosophila larver dendritiske arborization (da) neuroner er en populær model til undersøgelse mekanismer neuronal morfogenese. DA neuroner udvikle sig i forbindelse med de epidermale celler, de innerverer og dermed deres analyse fordele fra in situ visualisering af både neuronalt og epidermalt udtrykte proteiner ved immunfluorescens. Traditionelle metoder til fremstilling af larver fileter til immunofluorescens eksperimenter forlade intakt muskelvæv, der dækker det meste af kroppen væggen, præsentere en række udfordringer til billeddannelse neuronale og epidermale proteiner. Her beskriver vi en fremgangsmåde til fjernelse muskelvæv fra Drosophila larvernes fileter. Denne protokol muliggør billeddannelse af proteiner, som ellers skjules af muskelvæv, forbedrer signal-støj-forhold, og letter brugen af ​​super-opløsning mikroskopi for at studere da neuron udvikling.

Vi demonstrerer anvendeligheden af ​​proceduren for fjernelse musklen for at forbedre signal-støj-forholdet i immunofluorescens eksperimenter at samarbejde visualisere septate junction proteiner Coracle (Cora) og diske-store (Dlg) sammen med klasse IV da neuroner mærket med membranen markør CD4- tdTomato. Cora er tidligere blevet anvendt til at identificere skrifter hvorved da neuron dendritter er omsluttet af epidermale celler, og er en af mange ident...

Watch this Scientific Journal Video about Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells at JoVE.com

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Undersøgelser af neuronal morfogenese hjælp Drosophila larvestadiet dendritiske arborization (da) neuroner gavn af in situ visualisering af neuronale og epidermale proteiner ved immunofluorescens. Vi beskriver en fremgangsmåde, der forbedrer immunofluorescensanalyse af DA-neuroner og omgivende epidermisceller ved fjernelse muskelvæv fra larve kropsvæggen.

fjernelse af<em&gt; Drosophila</em&gt; Muskel Væv fra larver Filet for Immunofluorescens analyse af sensoriske neuroner og epidermale celler

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

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Neuroscience

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

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Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...