“<em>果蝇</em>视网膜是一小部分的刻板的方式所产生的细胞类型组成的类似晶体的晶格<sup> 1</sup>。其顺从先进的遗传分析,使研究复杂的发展计划。本协议描述了视网膜的解剖和免疫组化在三个连续的发育阶段,与光感受器分化的重点。
果蝇的复眼由约750小眼(单位只眼)。每个小眼组成的约20个细胞,包括镜头的分泌视锥细胞,色素细胞,刷毛细胞和8感光体(的PR)R 1-R 8 2。永久居民有专门的微绒毛的结构,rhabdomeres,其中含有光敏感的的颜料,Rhodopsins(右轴)。 6的PR(R1-R6)的rhabdomeres形成一个梯形和包含Rh1对3 4。 R7和R8的rhabdomeres串联在梯形的中心被定位在共享相同的路径的光。 R7和R8永久居民的随机表达不同的组合RHS在两个主要的亚型5的'p'亚型,RH3 在 p R7S加上RH5 在 p R8S,而在'Y'亚型,与RH4 在 y R7S RH6 在 y,R8S 6 7 8。
PR和发展,就要有规范的早期开始在幼虫眼触角成虫盘,单层的上皮细胞。差异化的浪潮席卷盘9和启动组件的未分化细胞,成小眼10-11。的创始人细胞的R8被指定为第,并招募R1-6和R7 12-14。随后,在蛹的发展,PR分化导致广泛的形态学改变,15日 ,,包括rhabdomere形成,突触形成和最终RH表达。
在这个协议中,我们描述了在三个定义的视网膜发育期,可应用于视网膜的形成和发展途径,以解决各种问题的视网膜解剖和免疫组化的方法。在这里,我们使用这些的方法可视化逐步PR分化,在单细胞水平在整个安装幼虫,midpupal和成人视网膜( <s豪华型>图1)。
1。故障排除
根据我们的经验,解剖需要实践(可长达数周),并促进实现舒适的手位21搁在桌子上,肘部和前臂和手指接触的解剖盘。这样一来,只有拇指,食指和中指进行细微的动作。
卸下椎板不损坏感光体的情况下可能是最具挑战性的步骤。红眼野生型的实践与飞白眼突变体解剖,第一层是对红色的眼睛色素沉着更容易看到。此外,使用灯光?…
The authors have nothing to disclose.
这项工作是由陆国际律师事务所奖学金HY支持。 H.,一个简棺童车纪念医学研究的博士后研究基金RJJ,美国国立卫生研究院资助F32EY016309到DV,纽约州立大学的教务长的论文奖学金DJ,NIH GrantR01 EY13010到CD和一个DFG奖学金JR(RI 2208/1- 1)。我们感谢龚如心Vogt和帕梅拉Boodram的手稿上的评论。
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. |