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 arbejde blev støttet af en Ehrman stipendium til HY. H., et Jane Coffin Childs Memorial Fund for Medical Research postdoc stipendium til RJJ, NIH Grant F32EY016309 til DV, en New York University Dean afhandling Fellowship til DJ, NIH GrantR01 EY13010 til CD og en DFG fællesskab til JR (RI 2208/1- 1). Vi takker Nina Vogt og Pamela Boodram om kommentarer til manuskriptet.

1. Indledning I denne video beskriver vi metoder til retinale dissektioner og immunhistokemi på tre definerede udviklingsmæssige perioder: den tredje stadiums larver, den midpupal og den voksne scene. Selvom vores protokol virker også på andre pupal faser (for detaljer om tidligere stadier, 16 se), valgte vi midpupal fase, da det er optimalt for billeddannelse alle PRs i ét brændplanet og deres kerner er let identificerbare, hvilket letter visualisering af transcriptionsfaktor...

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1. Fejlfinding Det er vores erfaring, kræver dissektioner praksis (op til flere uger), og lettes ved at opnå en komfortabel hånd position 21 ved at hvile albuerne og underarmene på bordet og med fingrene komme i kontakt med dissektion skålen. På den måde er det kun tommelfingre, indeks og midterste fingre udføre subtile bevægelser. Fjernelse af hinden uden at beskadige fotoreceptorerne er sandsynligvis den mest udfordrende trin. Praksis med rød-...

<table border="1"><tr><td></td><td></td><td></td><td> Reagens </td></tr><tr><td> Phosphatpufret saltvand (PBS1x, pH 7,4) </td><td> Sigma </td><td></td><td> Fremstille 10x stamopløsning 20. Fortyndes med destilleret vand til opnåelse af 1 x PBS og opbevares ved stuetemperatur. Afkøl på is før dissektioner. </td></tr><tr><td> Triton-X 100 </td><td> Sigma </td><td> 9002-93-1 </td><td> Forsigtig:! Lokalirriterende Brug handsker. Fremstilling af 50 ml 1X PBS med 0,3% Triton X-100 (PBST). Opbevares ved stuetemperatur. </td></tr><tr><td> 37% formaldehydopløsning </td><td> Fisher Scientific </td><td> F75P1GAL </td><td> Forsigtig:! Giftig, sandsynligvis et humant carcinogen Brug handsker. Før fiksationstrin, forberede frisk 3,7% opløsning i stinkskab ved fortynding med PBS, butik på is. </td></tr><tr><td> 5% normalt hesteserum </td><td> Jackson Immuno Research </td><td> 008-000-001 </td><td> Forbered 5% v / v fortynding i PBST. Opbevares ved fire grader. </td></tr><tr><td> Primære og sekundære antistoffer (f.eks æsel-anti-fåre-Alexa Fluor ® 488, Donkey-anti kanin-Alexa Fluor ® 555, Donkey anti-muse-Alexa Fluor ® 647) </td><td> Invitrogen Molecular Probes </td><td> A11015 A31572 A31571 </td><td> Fortynd sekundære antistoffer 1:800 i PBST og opbevar ved fire grader. </td></tr><tr><td> Alexa Fluor ® 488 phalloidin </td><td></td><td> A12379 </td><td> Fortyndes 1:100 i sekundær antistofopløsning. </td></tr><tr><td> Slowfade Gold antiblegemiddel reagens </td><td> Invitrogen Molecular Probes </td><td> S36936 </td><td> Monteringsmedium. Opbevar ved -20 grader. </td></tr><tr><td> Glycerol </td><td> Fisher Scientific </td><td> G31-1 </td><td> Til montering. Fremstilling af 10 ml af 50% fortynding med destilleret vand, opbevares ved stuetemperatur. </td></tr><tr><td> CO2 </td><td></td><td></td><td> Til bedøvelse af voksne fluer. </td></tr><tr><td></td><td></td><td></td><td> Udstyr </td></tr><tr><td> To skarpe pincet (Dumont # 55) </td><td> Fin Science Tools </td><td> 11.255-20 </td><td></td></tr><tr><td> Sylgard 184 silikoneelastomer Kit </td><td> Dow Corning </td><td></td><td> Forbered Sylgard dissektion skålen ved at fylde en plastik petriskål med Sylgard blanding. </td></tr><tr><td> Tre-brønds glas dissektion retter </td><td> Fisher Scientific </td><td> 21-379 </td><td></td></tr><tr><td> Two minutien dissekere stifter (0,1 mm diameter) og to pinholders (12 cm) </td><td> Fin Science værktøjer </td><td> 26.002-10 </td><td> Indsætte en minutien stift i hver af de to pinholders og bøj et af benene for at danne en krog. </td></tr><tr><td> Microscope dækglas (22x22-1 og 24x40-1) </td><td> Fisher Scientific </td><td> 12-542A </td><td></td></tr><tr><td> Objektglas (25x75x1.0 mm; precleaned) </td><td> Fisher Scientific </td><td> 12-550-143 </td><td></td></tr><tr><td> 1,5 ml mikrocentrifugerør </td><td></td><td></td><td></td></tr><tr><td> Klar neglelak eller Scotch tape </td><td></td><td></td><td></td></tr><tr><td> Rysteapparat </td><td> Bellco </td><td></td><td></td></tr><tr><td> Slide holder kasse </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> Tynd 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> Dissektionsmikroskop </td><td></td><td></td><td></td></tr><tr><td> Lille spand med is </td><td></td><td></td><td> Til køling af glas brøndsplader, dissekerede nethinde og løsninger. </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

Forbindelsen øje Drosophila melanogaster består af omkring 750 ommatidia (enhed øjne). Hver ommatidium er sammensat af omkring 20 celler, herunder linse-udskillende tapcellerne, pigment celler, en stritter celle og otte fotoreceptorer (PRS) R1-R8 2. PRS har specialiserede microvillar strukturer, rhabdomeres, der indeholder lysfølsomme pigmenter, de Rhodopsins (RHS). De rhabdomeres af seks PRS (R1-R6) danner en trapez og indeholder RH1 3 4. De rhabdomeres af R7 og R8 er anbragt efter hinanden i midten af ​​trapez og har samme vej lys. R7 og R8 PRs stokastisk udtrykker forskellige kombinationer af RHS i to undertyper 5: I &#39;p&#39; subtype, er RH3 i p R7s koblet med RH5 i p R8s, mens der i &#39;y&#39; subtype, er RH4 i y R7s forbundet med RH6 i y R8s 6 7 8. Tidlig specifikation af PRs og udvikling af ommatidia begynder i larvens eye-antennal imaginal disc, et monolag af epitelceller. En bølge af differentiering fejer hen over skiven 9 og initierer samling af udifferentierede celler i ommatidia 10-11. Den &#39;grundlægger celle&#39; R8 er angivet først og rekrutterer R1-6 og derefter R7 12-14. Efterfølgende under pupal udvikling, fører PR differentiering til omfattende morfologiske ændringer 15, herunder rhabdomere formation, synaptogenesen og til sidst rh ekspression. I denne protokol, beskriver vi metoder til retinale dissektioner og immunhistokemi på tre afgrænsede perioder nethinden udvikling, som kan anvendes til at løse en bred vifte af spørgsmål vedrørende retinal formation og udviklingsmæssige veje. Her bruger vi disse metoder til at visualisere den trinvise PR differentiering på enkelt-celle niveau i hele mount larve, midpupal og voksen nethinder ( <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; Retina er en krystal-lignende gitter bestående af et lille antal celletyper, der er frembragt i en stereotyp måde<sup&gt; 1</sup&gt;. Dens tilgængelighed for sofistikeret genetisk analyse tillader undersøgelse af komplekse udviklingsmæssige programmer. Denne protokol beskriver dissektioner og immunhistokemi af nethinder i tre diskrete udviklingsstadier, med fokus på fotoreceptor differentiering.

Dissektion og Immunhistokemi af Larvernes, Pupal og Voksen<em&gt; Drosophila</em&gt; Nethinder

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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