Dissektion og Immunhistokemi af Larvernes, Pupal og Voksen<em> Drosophila</em> Nethinder
Summary
Den<em> Drosophila</em> Retina er en krystal-lignende gitter bestående af et lille antal celletyper, der er frembragt i en stereotyp måde<sup> 1</sup>. 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.
Abstract
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 'p' subtype, er RH3 i p R7s koblet med RH5 i p R8s, mens der i 'y' 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 'grundlægger celle' 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> figur 1).
Protocol
Discussion
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-…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Reagent | |||
| Phosphate-buffered saline (PBS1x, pH 7.4) | Sigma | Prepare 10x stock solution 20. Dilute with distilled water to obtain 1x PBS and store at room temperature. Cool on ice before dissections. | |
| Triton-X 100 | Sigma | 9002-93-1 | Caution: Irritant! Wear gloves. Prepare 50 ml 1xPBS with 0.3% Triton X-100 (PBST). Store at room temperature. |
| 37% formaldehyde solution | Fisher Scientific | F75P1GAL | Caution: Toxic, probable human carcinogen! Wear gloves. Before the fixation step, freshly prepare 3.7% solution in a chemical fume hood by diluting with PBS, store on ice. |
| 5% normal horse serum | Jackson Immuno Research | 008-000-001 | Prepare 5% v/v dilution in PBST. Store at four degrees. |
| Primary and secondary antibodies (e.g. Donkey anti-sheep Alexa Fluor 488, Donkey-anti rabbit Alexa Fluor 555, Donkey anti-mouse Alexa Fluor 647) | Invitrogen Molecular Probes | A11015 A31572 A31571 |
Dilute secondary antibodies 1:800 in PBST and store at four degrees. |
| Alexa Fluor 488 Phalloidin | A12379 | Dilute 1:100 in secondary antibody solution. | |
| Slowfade Gold Antifade reagent | Invitrogen Molecular Probes | S36936 | Mounting medium. Store at -20 degrees. |
| Glycerol | Fisher Scientific | G31-1 | For mounting. Prepare 10 ml of 50% dilution with distilled water, store at room temp. |
| CO2 | For anesthetizing adult flies. | ||
| Equipment | |||
| Two sharp forceps (Dumont #55) | Fine Science Tools | 11255-20 | |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | Prepare Sylgard dissection dish by filling a plastic Petri dish with Sylgard mixture. | |
| Three-well glass dissection dishes | Fisher Scientific | 21-379 | |
| Two minutien dissecting pins (0.1 mm diameter) and two pinholders (12 cm) | Fine Science tools | 26002-10 | Insert one minutien pin in each of the two pinholders and bend one of the pins to form a hook. |
| Microscope cover slips (22×22-1 and 24×40-1) | Fisher Scientific | 12-542A | |
| Microscope slides (25x75x1.0 mm; precleaned) | Fisher Scientific | 12-550-143 | |
| 1.5 ml microcentrifuge tubes | |||
| Clear nail polish or Scotch tape | |||
| Orbital shaker | Bellco | ||
| Slide holder box | Fisher Scientific | ||
| Parafilm | Bemis | PM996 | |
| Thin paintbrush | |||
| P20 and P200 micropipettes and tips | |||
| Dissecting microscope | |||
| Small bucket with ice | For cooling the glass well plates, dissected retinas and solutions. |
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