3rd 龄 Drosophila 幼虫制备中幼虫节段神经的可视化

摘要

Drosophila melanogaster 幼虫为研究幼虫体节神经中轴突运输的机制提供了一个理想的模型系统。通过该实验流程，可将携带各种突变的3^rd 龄幼虫与野生型幼虫进行比较。

摘要

Drosophila melanogaster 正成为研究神经系统发育与功能的有力模型系统，尤其得益于其便利的遗传操作性和已完成全基因组测序的优势。此外，幼虫神经系统是研究轴突运输机制的理想模型系统，因为幼虫的体节神经包含成束的轴突，其胞体位于脑内，而神经末梢则沿身体长度分布。本文描述了在幼虫体节神经中可视化突触囊泡蛋白的操作流程。若操作得当，神经系统的所有组分以及相关组织（如肌肉和神经肌肉接头）均能保持完整、无损伤，并可用于后续观察。携带不同突变的3^rd 龄幼虫经解剖、固定、与突触囊泡抗体孵育后，与野生型幼虫一同进行观察和比较。该流程可适用于多种不同的突触或神经元抗体，便于在幼虫体节神经中直观观察多种蛋白分布的变化。

方案

1. Preparation of Reagents

1. Prepare 1x Dissection Buffer using 128 mM NaCl, 4mM CaCl₂, 4mM MgCl₂, 2 mM KCl, 5mM HEPES and 36mM Sucrose. PH the solution to 7.2 and filter sterilize.
2. Prepare the fixative using 1:1 dilution of 16% formaldehyde and 1x Dissection Buffer.
3. Prepare 1x PBT using 1x Phosphate Buffered Saline (PBS) at a PH of 7.2 and Triton X-100 in a 1:100 ratio.
4. Prepare 5% Bovine Serum Albumin (BSA) with 0.01% Na Azide in 1x PBS.

2. Preparation for the Dissection

1. Sylgard dishes are used for dissection. Prepare the dishes by pouring Sylgard 184 silicone elastomer base and curing agent mixture into a Petri dish. Allow the mixture to completely solidify and dry before using.
2. Before the dissection, gather a clean pair of #5 forceps, a clean pair of straight blade vannas scissors, and pins.

3. Dissection of 3^rd Instar Larvae

1. Collect wandering 3^rd instar larvae of a specific genotype onto a petri dish.
2. Wash the larvae in deionized water to get rid of the food.
3. Place a larva on the sylgard dish, with the dorsal side up. Pin the larva at its anterior and posterior ends.
4. Place a drop of 1x Dissection Buffer on the pinned larva. Using fine scissors nip the larva on the dorsal midline near the posterior end. Using the scissors, cut the larval cuticle from the posterior end through to the anterior end.
5. Place a drop of new 1x Dissection Buffer onto the dissected larva. Using a pin, pick one lateral edge of the cuticle near the middle of the larva and pin the cuticle. Using another pin, pin the second lateral edge of the cuticle as shown in the diagram. Two pins are used on each lateral side. See diagram below.
6. Carefully remove the intestines, fat bodies, and trachea, leaving only the brain with the two optical lobes and ventral ganglia with the segmental nerves attached. The remainder of the procedure is done on the sylgard dish.

4. Fixation of the Dissected Larva

1. Incubate the dissected larva in fix for 30 minutes, by replacing the fix every 15 minutes.
2. After fixation, rinse the larva in 1x PBT for 30 minutes by replacing the buffer every 10 minutes. It is important to note that the larva should always be in a buffer.

5. Antibody Staining of the Dissected Larva

1. Prepare the primary antibody by diluting it to the appropriate concentration in 5% BSA with Na Azide in 1x PBT. Optimal concentrations will differ for different antibodies.
2. Replace the last 1x PBT rinse with the prepared primary antibody solution, and incubate in a humid chamber at 4°C overnight. The humid chamber is constructed by lining a plastic dish with kim-wipes soaked in water.
3. The following day, rinse the dissected larva in 1x PBT for 30 minutes by replacing the buffer every 10 minutes.
4. Prepare the secondary antibody by diluting it to the appropriate concentration in 5% BSA with Na Azide in 1x PBT, and incubate the larva with the secondary antibody solution at 25°C for an hour. Wrap the humid chamber in foil to prevent light, as secondary antibodies are light sensitive.
5. After incubation, rinse the larva in 1x PBT for 30 minutes by replacing the buffer every 10 minutes.

6. Mounting and Visualization of the Dissected Larva

1. Before mounting the larva on the slide, prepare the slide by spreading two vertical lines of nail polish in the middle, with a space enough for a cover slip to sit on (see diagram). Let the nail polish dry completely.
2. Place a drop of mounting buffer in the space between the vertical nail polish lines on the slide (see diagram). The slide is now ready for mounting.
   
   
3. After the last 1x PBT rinse, gently remove the lateral pins. Be careful to not tear the cuticle.
4. Carefully remove the two remaining pins and pick up the larva with forceps and place the larva horizontally on the prepared slide. Make sure the larva is not flipped over and is oriented ventral side up.
5. Gently place a cover slip on top and seal the edges with nail polish (see diagram). The larva is now ready for visualization under a fluorescent microscope.
   

7. Representative Results

Figure 1: A representative image of larval segmental nerves from a wild type larva using antibodies against the synaptic vesicle protein cysteine string protein (CSP, Zinsmaier et al.1994 ). Note that the segmental nerves are smoothly stained. Bar=20μm

Figure 2: A representative image of larval segmental nerves from a motor protein mutant larva using antibodies against the synaptic vesicle protein cysteine string protein (CSP). Note that the segmental nerves show massive accumulations that stain brightly for CSP (arrows).

讨论

该 果蝇 幼虫节段神经是研究轴突运输机制的有力系统。如果3^rd 若正确进行幼虫龄期解剖，可在单个幼虫中观察中枢神经系统（CNS）、外周神经系统（PNS）以及相关组织（如肌肉和神经肌肉接头）。本实验方案改编自Hurd和Saxton（1996）发表的方法。该方案亦可适用于其他神经元抗体的检测，但需对抗体条件进行优化。一抗和二抗的孵育时间可作调整，也可增加封闭步骤。我们已成功应用此方案研究淀粉样前体蛋白的功能 ¹ 和亨廷顿蛋白 ² 在轴突运输中。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
Dumont #5 镊子	精细科学工具公司	11252-20
微型不锈钢固定针	精细科学工具公司	26002-10
Mcpherson-Vannas 直刃剪刀，8 cm	费舍尔科技公司	50822236
Vectashield 封片介质	Vector Laboratories 公司	H-1000
Sylgard 184 硅橡胶弹性体	道康宁公司	4026148
甲醛，16%	电子显微镜科学公司	15710
dCSP3	发育研究杂交瘤库
Alexa Fluor 568 山羊抗小鼠 IgG	英杰公司	A-11004