Name: dissection and immunohistochemistry of larval, pupal and adult drosophila retinas
Uploaded: 2012-11-14T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dette arbeidet ble støttet av en Ehrman fellesskap å HY. H., en Jane Coffin Childs minnefond for Medical Research postdoktorstipend til RJJ, NIH Grant F32EY016309 til DV, en New York University Dean Dissertation Fellowship til DJ, GrantR01 EY13010 NIH til CD og en DFG fellesskap å JR (RI 2208/1- 1). Vi takker Nina Vogt og Pamela Boodram for kommentarer til manuskriptet.

1. Innledning I denne videoen beskriver vi metoder for netthinnens disseksjoner og immunhistokjemi på tre definerte utviklingsmål perioder: den tredje instar larver, den midpupal og det voksne stadiet. Selv om vår protokoll fungerer også for andre pupal stadier (for detaljer om tidligere stadier, se 16), valgte vi midpupal scenen, som det er optimalt for bildebehandling alle PRs i ett fokusplan og deres kjerner er lett identifiserbar, noe som letter visualisering av transkripsjo...

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1. Feilsøking I vår erfaring, disseksjoner krever praksis (opptil flere uker) og er tilrettelagt av å oppnå en komfortabel håndstilling 21 ved hvile albuene og underarmene på bordet, og med fingrene å ta kontakt med disseksjon parabolen. På den måten, bare tommelen, indeksen og midtre fingrene utføre subtile bevegelser. Fjerne lamina uten å skade fotoreseptorene er trolig den mest utfordrende trinnet. Praksis med rød-eyed wildtype flyr først ...

<table border="1"><tr><td></td><td></td><td></td><td> Reagens </td></tr><tr><td> Fosfat-bufret saltvann (PBS1x, pH 7,4) </td><td> Sigma </td><td></td><td> Forbered 10x stamløsning 20. Fortynn med destillert vann for å få 1x PBS og oppbevar ved romtemperatur. Avkjøl på isen før disseksjoner. </td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Forsiktig: Irriterende Bruk hansker. Forbered 50 ml 1xPBS med 0,3% Triton X-100 (PBST). Oppbevar ved romtemperatur. </td></tr><tr><td> 37% formaldehydoppløsning </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Forsiktig: Giftig, sannsynlig kreftfremkallende stoff Bruk hansker. Før fiksering trinnet tilbereder 3,7% løsning i avtrekkskap ved fortynning med PBS, lagre på is. </td></tr><tr><td> 5% normal hest serum </td><td> Jackson Immuno Forskning </td><td> 008-000-001 </td><td> Forbered 5% v / v fortynning i PBST. Lagre på fire grader. </td></tr><tr><td> Primære og sekundære antistoffer (f.eks Donkey anti-sau Alexa 488 Fluor, Donkey-antikanin Alexa Fluor 555, Donkey anti-mus Alexa Fluor 647) </td><td> Invitrogen Molecular Probes </td><td> A11015 A31572 A31571 </td><td> Fortynne sekundære antistoffer 1:800 i PBST og butikken på fire grader. </td></tr><tr><td> Alexa Fluor 488 phalloidin </td><td></td><td> A12379 </td><td> Fortynne 1:100 i sekundært antistoff løsning. </td></tr><tr><td> Slowfade Gold Antifade reagens </td><td> Invitrogen Molecular Probes </td><td> S36936 </td><td> Montering medium. Oppbevar ved -20 grader. </td></tr><tr><td> Glycerol </td><td> Fisher Scientific </td><td> G31-1 </td><td> For montering. Forbered 10 ml 50% fortynning med destillert vann, oppbevares ved romtemperatur. </td></tr><tr><td> CO 2 </td><td></td><td></td><td> For bedøvelse voksne fluer. </td></tr><tr><td></td><td></td><td></td><td> Utstyr </td></tr><tr><td> To skarpe tang (Dumont # 55) </td><td> Fin Science Verktøy </td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard 184 Silikon Elastomer Kit </td><td> Dow Corning </td><td></td><td> Forbered Sylgard disseksjon parabolen ved å fylle en plast petriskål med Sylgard blanding. </td></tr><tr><td> Tre brønner glass disseksjon retter </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> Two minutien dissecting pins (0,1 mm diameter) og to pinholders (12 cm) </td><td> Fin Science verktøy </td><td> 26002-10 </td><td> Sett en minutien pin i hver av de to pinholders og bøy en av pinnene for å danne en krok. </td></tr><tr><td> Microscope dekkglass (22x22-1 og 24x40-1) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Mikroskopobjektglass (25x75x1.0 mm; precleaned) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> 1,5 ml mikrosentrifugerør </td><td></td><td></td><td></td></tr><tr><td> Klar neglelakk eller Scotch tape </td><td></td><td></td><td></td></tr><tr><td> Orbital shaker </td><td> Bellco </td><td></td><td></td></tr><tr><td> Lysbildeholder boks </td&gt; <td> Fisher Scientific </td><td></td><td></td></tr><tr><td> Parafilm </td><td> Bemis </td><td> PM996 </td><td></td></tr><tr><td> Tynn pensel </td><td></td><td></td><td></td></tr><tr><td> P20 og P200 mikropipetter og tips </td><td></td><td></td><td></td></tr><tr><td> Disseksjonsmikroskop </td><td></td><td></td><td></td></tr><tr><td> Liten bøtte med is </td><td></td><td></td><td> For kjøling av glass brønns plater, dissekert netthinne og løsninger. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

Den sammensatte øye Drosophila melanogaster består av ca 750 ommatidia (enhet øyne). Hver ommatidium består av ca 20 celler, inkludert objektiv-sekresjon membran celler, pigment celler, en bust celle og åtte fotoreseptorene (PRS) R1-R8 to. PRS har spesialisert microvillar strukturer, de rhabdomeres, som inneholder lysfølsomme pigmenter, de Rhodopsins (RHS). De rhabdomeres av seks PRS (R1-R6) danner et trapes og inneholder RH1 3 4. De rhabdomeres av R7 og R8 er plassert i tandem i sentrum av trapezoid og dele den samme banen av lys. R7 og R8 PRs stokastisk uttrykker ulike kombinasjoner av RHS i to undergrupper 5: I &quot;p&quot; undertype, er RH3 i p R7s kombinert med RH5 i p R8s, mens i &quot;y&quot; undertype, er RH4 i y R7s forbundet med RH6 i y R8s 6 7 8. Tidlig spesifikasjon av PRs og utvikling av ommatidia begynner i larve øye-antennal imaginal plate, en monolayer av epitelceller. En bølge av differensiering feier over disk 9 og initierer montering av udifferensierte celler til ommatidia 10-11. The &#39;grunnlegger celle&#39; R8 er spesifisert først og rekrutterer R1-6 og deretter R7 12-14. Deretter, i løpet pupal utvikling, fører PR differensiering til omfattende morfologiske endringer 15, inkludert rhabdomere formasjon, synaptogenesis og til slutt rh uttrykk. I denne protokollen beskriver vi metoder for netthinnens disseksjoner og immunhistokjemi på tre definerte perioder med netthinnen utvikling, som kan brukes til å løse en rekke spørsmål om retinal formasjon og utviklingsmessige veier. Her bruker vi disse metodene for å visualisere en trinnvis PR differensiering på enkelt-celle-nivå i hele mount larve, midpupal og voksen netthinne ( <sTrong&gt; Figur 1).

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Den<em&gt; Drosophila</em&gt; Netthinnen er en krystall-lignende gitter sammensatt av et lite antall av celletyper som er generert i en stereotyp måte<sup&gt; 1</sup&gt;. Sin amenability til sofistikert genetisk analyse gjør studiet av komplekse utviklingsprogrammene. Denne protokollen beskriver disseksjoner og immunhistokjemi av netthinne på tre diskrete utviklingsstadier, med fokus på fotoreseptoren differensiering.

Disseksjon og Immunhistokjemi av larver, pupal og Voksen<em&gt; Drosophila</em&gt; Netthinne

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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