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Open-book Preparations from Chick Embryos and DiI Labeling of Commissural Axons
雏鸡胚胎的开放式制备和合缝轴突的DiI标记

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Abstract

Successful neural circuit formation relies on the accurate navigation of axons towards their targets during development. Axons are guided by a combination of short-range and long-range, attractive and repulsive cues. The commissural axons of the developing spinal cord have provided an informative in vivo model for the identification of multiple axon guidance molecules and mechanisms. These axons extend ventrally from the dorsal spinal cord and cross the midline at the floor plate, before making a sharp rostral turn towards the head. This simple trajectory has facilitated the identification of many axon guidance molecules, because perturbation of the stereotypical guidance decisions as a result of genetic manipulations can be easily identified. The open-book assay is a method to assess the trajectory of spinal commissural axons. The spinal cord is dissected out, opened at the roof plate and pinned flat. Punctate injections of the lipophilic fluorescent dye, DiI, are used to trace commissural axon trajectories prior to microscopy and analysis.

Keywords: Axon guidance(轴突导向), Spinal cord(脊髓), Neural development(神经发育), Dissection(夹层), Axon tracing(轴突追踪)

Materials and Reagents

  1. Fertilized eggs containing chicken embryos (incubated until Hamburger and Hamilton stage 25-26)
  2. PBS (add 50 ml of 20x PBS to 950 ml of ddH2O)
  3. Sylgard silicone elastomer (World Precision Instruments, catalog number: SYLG184 )
  4. Fast DiI (Life Technologies, Molecular Probes®, catalog number: D-7756 ) (dissolved at 5 mg/ml in ethanol)
  5. Vacuum grease (Dow Corning Corporation, catalog number: 976V )
  6. 20x sterile phosphate buffered saline (PBS) (see Recipes)
  7. 4% paraformaldehyde (4% PFA) (see Recipes)
  8. Phosphate (PB) buffer (1 M, 1 L, pH 7.4) (see Recipes)

Equipment

  1. Incubator set at 38.5 °C and 45% humidity (e.g. FIEM, Juppiter 576 Setter+Hatcher; Kendro Laboratory Products, Heraeus/Kendro, model: B12 )
  2. Spring scissors (Fine Science Tools, catalog number: 15003-08 )
  3. Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
  4. 90 mm Petri dishes (e.g. Thermo Fisher Scientific, Sterilin®, catalog number: 101VR20/C )
  5. 0.20 mm insect pins (Fine Science Tools, catalog number: 26002-20 )
  6. 0.10 mm insect pins (Fine Science Tools, catalog number: 26002-10 )
  7. Microscope with adjustable transmitted light source (Leica MZ6 stereomicroscope with Leica CLS 150x )
  8. Fine surgical scalpel (Grieshaber Logistic Group, catalog number: 68101 )
  9. Tungsten wire (0.075 mm) (World Precision Instruments, catalog number: TGW0325 )
  10. Metal flat spatula/spoon (e.g. Sigma-Aldrich, catalog number: S4022 , S3397 )
  11. Borosilicate glass capillaries (outer Ø/inner Ø: 1.2 mm/0.68 mm) (World Precision Instruments, catalog number: 1B120F-4 )
  12. Glass needle puller (Narishige Group, catalog number: PC-10 )
  13. Polyethylene tubing (Ø 1.24 mm) (e.g. Angst and Pfister AG, catalog number: FT0110350018 )
  14. Plastic transfer pipettes (e.g. Sarstedt, catalog number: 86.1171 )
  15. 18 G x 1.5” needle (e.g. Braun, catalog number: 4665120 )
  16. 1 ml syringe (e.g. Braun, catalog number: 9166017V )
  17. 20 ml syringe (e.g. Braun, catalog number : 4606205V )
  18. 24 mm x 24 mm glass coverslips (VWR International, catalog number: 631-0127 )
  19. Microscope suitable for brightfield and fluorescent microscopy (e.g. Olympus, model: BX51 )

Procedure

  1. Preparation of open-books
    1. PBS is added to a Petri dish lined with ~5 mm of Sylgard elastomer (prepared previously, following the manufacturer’s instructions).
      Note: Sylgard is required as a substrate for the insect pins during the dissection. Petri dishes lined with Sylgard can be made in advance (in batches) and reused for several years.
    2. At HH25-26 (day 5 of development), the chicken embryo is removed from the egg using forceps and a small spatula and placed in the Petri dish (Figure 1a).
      Note: For windowed eggs (experimentally manipulated), the embryo can be carefully lifted out of the egg through the window by inserting a closed pair of forceps under the neck. Non-windowed eggs should be carefully cracked into a dish. The embryo can be separated from the blood vessels using forceps and spring scissors, rinsed in PBS, then transferred to the dissection dish.
    3. The extraembryonic membranes are removed with forceps and the head is cut off.
      Note: The embryo can be stabilized during this step if necessary, by inserting a 0.2 mm insect pin through the eye. Remove the membranes by grasping them with forceps under the neck, then gently pull them over the head and down towards the tail.
    4. The embryo is placed on its back and pinned (using 0.2 mm insect pins) through the neck and tail, with gentle stretching.
    5. The embryo is pinned (using 0.2 mm insect pins) through the wing buds and limb buds (Figure 1b).
      Note: All pins should be inserted at an angle away from the embryo so that they do not interfere with the next steps of the dissection.
    6. The heart and internal organs are removed using spring scissors and gently scraping with forceps.
      Note: The embryo should be illuminated from below to enable tissue density to be perceived. The segmented vertebrae should be visible if all the organs have been removed completely (Figure 1c).
    7. Using spring scissors, a shallow cut is made through the vertebrae overlying the spinal cord at the neck, and longitudinally for a few vertebral segments along each side, towards the tail (Figure 1c).
      Note: Do not cut too deeply or too close to the spinal cord. Rotate the dish to make the cutting angle easier.
    8. Forceps are used to grasp and lift the flap of vertebrae away from the spinal cord. With gentle pulling towards the tail, the vertebrae overlying the spinal cord are removed in a single strip of tissue (Figure 1d).
      Note: Be careful not to damage the spinal cord.
    9. The embryo is gently stretched and re-pinned through the tail and limbs.
    10. A fine microsurgical scalpel (or a tungsten needle) is used to cut away the meninges overlying the spinal cord.
      Note: Look for a denser line of tissue between the spinal cord and the dorsal root ganglia. Adjust the illumination angle, if necessary (Figure 1e). Gently cut longitudinally along this line on each side of the spinal cord, from the neck to the tail. The meninges should separate from the spinal cord due to the gentle stretching of the pinned embryo (Figure 1f).
    11. Using spring scissors, the spinal cord is cut at the level of the wing buds and caudal to the limb buds.
    12. Using forceps, the isolated spinal cord is grasped at the rostral end and removed from the embryo in a single piece, by gently pulling it in a smooth motion towards the tail (Figure 1g).
      Note: The spinal cord is kept immersed in PBS during this step. In chickens, a small amount of the dorsal spinal cord (the roof plate) is left behind. The isolated spinal cord will therefore resemble a sheet rather than a tube.
    13. The isolated spinal cord is gently lifted onto a flat metal spatula (for support) and transferred to a new Sylgard-lined Petri dish containing 4% paraformaldehyde in PBS.
      Note: The spinal cord is very delicate and should be gently immersed in the 4% PFA using closed forceps (or another blunt tool). Avoid touching the spinal cord at all, if possible. The 4% PFA solution should be at room temperature, as the spinal cords tend to float if the 4% PFA is cold. To break the surface tension, it is also possible to place 1-2 of the previously dissected heads in the dish of 4% PFA, prior to transferring the spinal cords.
    14. A flat-mount preparation (Figure 2) is produced by carefully pinning the spinal cord in six positions (rostrally, medially and caudally on each side), using 0.10 mm insect pins (Figure 1h). The medial surface of the spinal cord (which previously lined the central canal of the closed neural tube) should be facing upwards.
      Note: Angle the pins away from the spinal cord to prevent them interfering with the subsequent DiI injections. We label each open-book using a little ‘flag’ (made of tape wrapped around a 0.2 mm insect pin) that not only identifies the embryo from which the open-book was derived, but also indicates the anterior end of the preparation.
    15. The open-book preparations are fixed in 4% PFA for 30 min to 1 h at room temperature.
      Note: Open-books should not be over-fixed as this reduces the efficiency of DiI diffusion and increases background.
    16. The fixative is carefully poured out of the dish and replaced with PBS.
      Note: Preparations should be refrigerated until ready to inject with DiI or mount for microscopic inspection. DiI should be injected within 2 weeks.

  2. Injection of open-book preparations with DiI
    1. A needle puller is used to make glass micropipettes from borosilicate glass capillaries.
    2. A glass micropipette is inserted into polyethylene tubing.
    3. Using forceps, the end of the micropipette is broken off, to obtain a very small diameter opening (~5 μm) at the tip.
    4. Fast DiI (5 mg/ml in ethanol) is drawn into the micropipette using gentle mouth suction.
      Note: Do not draw up too much DiI, in case the tip of the needle breaks during the injection procedure, causing the DiI to spill and ruin your samples. Insert the needle into a dish of PBS and gently blow some out. Check that the DiI does not continue to leak from the needle after you have finished blowing. If the DiI leaks, the needle diameter is too big. In that case, prepare a new needle.
    5. The open-book preparations are illuminated from below, and the region to be injected is identified.
      Note: The cell bodies of the commissural neurons are located just ventral to the roof plate and just dorsal to the dorsal funiculus. The dorsal funiculus can be identified in the open-books due to its different density compared to the surrounding tissue. Look for a denser longitudinal stripe of tissue, located approximately 1/5 of the width of a hemibook from the lateral edge of the preparation (Figure 1i). The commissural neurons are located immediately lateral to this stripe (i.e. immediately dorsal to the dorsal funiculus). Make your injections along the lateral border of the dorsal funiculus.
    6. Starting at one end of the open-book, the glass needle is inserted into the tissue at an angle of >45° and, as the needle is withdrawn, a small amount of DiI is gently blown in.
      Note: Because ethanol is less dense than PBS, the DiI will float away from the injection site, which may impede your view. Try not to inject excess DiI. You may need to practise mouth-pipetting to get this right.
    7. Working quickly, DiI injections are made along the length of the open-book at regular intervals of approximately 0.5 mm (Figure 1j).
      Note: If the needle becomes clogged, carefully clear it with forceps. If the tip becomes too big (and DiI leaks), replace the needle.
    8. After completing the injections of each open-book, a transfer pipette is used to suck away and discard any excess DiI.
      Note: Failure to do this will result in high background.
    9. The injected open-book preparations are refridgerated for 3-4 days to allow the DiI to diffuse along the axons.
      Note: If necessary, top up the Petri dish with PBS to ensure that the preparations remain immersed during the DiI diffusion.

  3. Mounting and microscopic analysis
    1. A 1 ml syringe is filled with vacuum grease and fitted with an 18 G needle.
    2. The syringe is used to spread a thin, uninterrupted border of vacuum grease around the edges of a 24 mm x 24 mm glass coverslip.
      Note: The grease layer should not be too thick, otherwise it will be difficult to visualize the preparations under microscopy. Make sure there are no gaps in the grease border, or the preparations will dry out. The type of grease used is also important. Vacuum grease of low viscosity may mix with the PBS (in subsequent steps) and result in high background.
    3. Several drops of sterile PBS are added to the middle of the well.
      Note: Mounting medium containing glycerol with n-propyl gallate may not be compatible with DiI.
    4. The pins are removed from the open-book and it is transferred into the PBS droplet using forceps.
    5. The open-book is immersed in the PBS and positioned in the middle of the well.
    6. Another 24 mm x 24 mm coverslip is gently placed on top, making sure the open-book stays open and there are no significant air bubbles.
      Note: If necessary, the coverslip can be removed, and the open-book repositioned.
    7. Gentle pressure is applied to the edges of the coverslips to create a complete seal of grease.
      Note: Excess PBS will be squeezed out during this step. Again, avoid air bubbles.
    8. The preparations are kept refrigerated in the dark until ready for inspection and documentation by fluorescent microscopy (Figure 1k-l). Mounted preparations are placed on top of a glass microscope slide for imaging and analysis.
      Note: The PBS will eventually dry out. Analysis should be done within 1-2 weeks, to ensure that the preparations do not dry out before being assessed.


      Figure 1. Steps in open-book preparation

      1.  HH25-26 chicken embryo in a dish of PBS. The extraembryonic membranes have been removed.
      2. Embryo (with head removed) is pinned through the neck, tail, wings and limbs. Pins are angled away from the body. Anterior is to the left.
      3. Embryo with the heart and organs removed. The segmented vertebrae are now visible. The area to be cut by the spring scissors to create a flap of vertebral tissue is shown by the dotted lines.
      4. Some of the vertebrae at the anterior end have been removed, exposing the spinal cord. At the posterior end, the vertebrae are still overlying the spinal cord.
      5. Exposed spinal cord with all vertebrae removed. The DRGs are visible. Arrows indicate the dense line of tissue where longitudinal cuts should be made to separate the meninges from the spinal cord.
      6. Zoomed views of preparation before and after the spinal meninges (arrows) have been cut.
      7. Preparation after the spinal cord has been lifted out of the embryo.
      8. Isolated spinal cord, pinned into the flat ‘open-book’ configuration (see Figure 2). The floor plate lies medially and the dorsal part of the spinal cord is now located laterally. Pins located rostrally (R) medially (M) and caudally (C) are angled away from the preparation.
      9. In higher magnification, the dense longitudinal stripe of tissue corresponding to the dorsal funiculus is indicated by arrows. DiI injections should be made along the lateral border of the dorsal funiculus.
      10. Brightfield image of a mounted open-book preparation with DiI injection sites (red).
      11. Fluorescent image of an open-book preparation with DiI injection sites (red). The commissural axons, which have grown ventrally and crossed the floor plate, can be seen.
      12. Higher magnification of a single DiI injection site. Commissural axons grow towards and across the floor plate before turning anteriorly.


        Figure 2. Schematic overview of procedure. The spinal cord is dissected out, opened dorsally at the roofplate and laid flat. The commissural neurons (CN, shown in blue) extend axons that grow ventrally and cross the floor plate (FP) before turning anteriorly. Commissural axons are labeled by injections of DiI (red). a, anterior; p, posterior; d, dorsal; v, ventral.

Notes

  1. The isolated spinal cords are very fragile. Avoid touching them as much as possible and minimize/avoid changes of solution. When transferring the spinal cords from dish to dish, or when mounting the DiI-injected open-books for analysis, always grasp them at the anterior end using forceps. That way, only a small part of the spinal cord will be damaged during the handling.
  2. We have previously published a video protocol demonstrating electroporations, open-book dissections and DiI injection steps, which may be useful (Wilson and Stoeckli, 2012).
  3. For further details of chicken embryo incubation and handling, see Reference 1 or Reference 4.
  4. Please refer to Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation (Ito-Ishida, 2013) for mouth pipetting approaches.

Recipes

  1. 20x sterile PBS (1 L)
    160 g of NaCl
    4 g of KCl
    28.84 g of Na2HPO4.2H2O
    4.14 g of NaH2PO4.H2O
    Dissolved in 1 L ddH2O, autoclave.
  2. 4% paraformaldehyde (4% PFA) (200 ml)
    150 ml of ddH2O
    0.8 ml of 2 M NaOH
    8 g of Paraformaldehyde
    Heat at 70 °C for 30 min, cool, add 20 ml of 1 M PB buffer.
    Adjust pH to 7.4, make up to 200 ml with ddH2O.
    Stored at 4 °C
  3. Phosphate (PB) buffer (1 M, 1 L, pH 7.4)
    206.6 g of Na2HPO4.7H2O
    32 g of NaH2PO4.H2O
    Dissolved in 1 L ddH2O

Acknowledgments

This protocol was adapted from the previously published papers Wilson and Stoeckli (2012) and Andermatt et al. (2014). This work was supported by a grant from the Swiss National Science Foundation to E.S.

References

  1. Andermatt, I., Wilson, N. and Stoeckli, E. T. (2014). In ovo electroporation of miRNA-based-plasmids to investigate gene function in the developing neural tube. Methods Mol Biol 1101: 353-368.
  2. Ito-Ishida, A. (2013). Labeling of precursor granule cells in the cerebellum by ex vivo electroporation. Bio-protocol 3(12): e778.
  3. Wilson, N. H. and Stoeckli, E. T. (2013). Sonic hedgehog regulates its own receptor on postcrossing commissural axons in a glypican1-dependent manner. Neuron 79(3): 478-491.
  4. Wilson, N.H. and Stoeckli, E. T. (2012). In ovo electroporation of miRNA-based plasmids in the developing neural tube and assessment of phenotypes by dil injection in open-book preparations. J Vis Exp (68), e4384, doi:10.3791/4384.

简介

成功的神经电路形成依赖于在发展期间轴突朝向其靶标的精确导航。轴突由短程和长程,吸引和排斥线索的组合引导。发育中的脊髓的连合轴突提供了用于多轴突导向分子和机制的鉴定的信息的体内模型。这些轴突从背侧脊髓向腹侧延伸并在底板处穿过中线,然后朝向头部进行尖锐的嘴唇转向。这个简单的轨迹有助于许多轴突指导分子的识别,因为可以容易地识别作为遗传操作的结果的定型指导决定的干扰。开放书测定是评估脊柱连合轴突的轨迹的方法。脊髓被解剖出来​​,在屋顶板上打开并固定平。亲脂性荧光染料DiI的点状注射用于在显微镜和分析之前追踪连合轴突轨迹。

关键字:轴突导向, 脊髓, 神经发育, 夹层, 轴突追踪

材料和试剂

  1. 含有鸡胚的受精卵(培养至汉堡和汉密尔顿25-26级)
  2. PBS(加入50ml 20x PBS至950ml ddH 2 O)中
  3. Sylgard有机硅弹性体(World Precision Instruments,目录号:SYLG184)
  4. 快速DiI(Life Technologies,Molecular Probes ,目录号:D-7756)(以5mg/ml溶解于乙醇中)
  5. 真空脂(Dow Corning Corporation,目录号:976V)
  6. 20x无菌磷酸盐缓冲盐水(PBS)(参见Recipes)
  7. 4%多聚甲醛(4%PFA)(参见配方)
  8. 磷酸盐(PB)缓冲液(1M,1L,pH7.4)(参见配方)

设备

  1. 设置在38.5℃和45%湿度(例如FIEM,Juppiter 576Sterter + Hatcher; Kendro Laboratory Products,Heraeus/Kendro,型号:B12)的孵育器
  2. 弹簧剪(Fine Science Tools,目录号:15003-08)
  3. Dumont#5镊子(Fine Science Tools,目录号:11252-20)
  4. 90mm培养皿(例如Thermo Fisher Scientific,Sterilin ,目录号:101VR20/C)。
  5. 0.20毫米昆虫针(Fine Science Tools,目录号:26002-20)
  6. 0.10mm昆虫针(Fine Science Tools,目录号:26002-10)
  7. 具有可调透射光源的显微镜(Leica MZ6立体显微镜,配有Leica CLS 150x)
  8. 美容手术刀(Grieshaber Logistic Group,目录号:68101)
  9. 钨丝(0.075mm)(World Precision Instruments,目录号:TGW0325)
  10. 金属平刮刀/勺子(例如Sigma-Aldrich,目录号:S4022,S3397)
  11. 硼硅玻璃毛细管(外径Ø/内径:1.2 mm/0.68 mm)(World Precision Instruments,目录号:1B120F-4)
  12. 玻璃拔针器(Narishige集团,目录号:PC-10)
  13. 聚乙烯管(Ø1.24mm)(例如Angst和Pfister AG,目录号:FT0110350018)
  14. 塑料移液管(如 Sarstedt,目录号:86.1171)
  15. 18 G x 1.5"针(例如Braun,目录号:4665120)
  16. 1ml注射器(例如Braun,目录号:9166017V)
  17. 20ml注射器(例如Braun,目录号:4606205V)
  18. 24mm×24mm玻璃盖玻片(VWR International,目录号:631-0127)
  19. 适用于明场和荧光显微镜(例如 Olympus,型号:BX51)的显微镜

程序

  1. 准备开放书
    1. 将PBS加入衬有〜5mm Sylgard弹性体的培养皿中 (按照制造商的说明书预先制备)。
      注意:   需要Sylgard作为昆虫针的基质 解剖。 内衬Sylgard的培养皿可提前提取(in 批次),并重复使用几年。
    2. 在HH25-26(第5天 发育),使用镊子从鸡蛋中取出鸡胚 和一个小铲子,并放置在培养皿(图1a) 注意: 对于窗口卵(实验操纵),胚胎可以 通过插入一个封闭的,通过窗口小心地提出了蛋   对镊子在脖子下。 非窗口鸡蛋应该仔细 破裂成一个盘。 胚胎可以与血管分离 使用镊子和春天剪刀,在PBS中漂洗,然后转移到 解剖碟。
    3. 用镊子取出胚外膜,并将头部切掉 注意:   如果需要,胚胎可以在该步骤期间稳定 插入0.2毫米的昆虫针通过眼睛。 通过去除膜 用镊子抓住他们,然后轻轻地拉他们 头部和尾部。
    4. 将胚胎放置在其背部并通过颈部和尾部固定(使用0.2mm昆虫针),并轻柔地拉伸。
    5. 胚胎通过翼芽和肢芽固定(使用0.2mm昆虫针)(图1b)。
      注意:   所有的销应该以一定的角度离开胚胎插入 它们不会干扰解剖的后续步骤。
    6. 使用弹簧剪刀和用镊子轻轻刮去心脏和内脏 注意:   胚胎应从下面照亮,以使组织密度   被感知。 分割的椎骨应该是可见的,如果所有 器官已完全清除(图1c)。
    7. 使用弹簧 剪刀,浅切割通过椎骨覆盖 脊髓在颈部,纵向为几个椎骨节段   沿着每一侧,朝向尾巴(图1c) 注意:不要切割太深或太靠近脊髓。 旋转培养皿,使切割角更容易。
    8. 钳子用于抓住和提起椎骨的瓣片远离   脊髓。 轻轻拉向尾部,椎骨 覆盖脊髓在单个条带组织中被去除 (图1d)。
      注意:小心不要损伤脊髓。
    9. 胚胎轻轻地伸展,并通过尾部和四肢固定
    10. 使用精细的显微外科手术刀(或钨针)切除覆盖脊髓的脑膜。
      注意:   寻找脊髓和背部之间的更密集的组织线   根神经节。 如有必要,调整照明角度(图1e)。 在脊柱的每一侧沿着这条线沿纵向轻轻切开 线,从颈部到尾部。 脑膜应该分开 脊髓由于固定胚胎的温和拉伸(图 1f)。
    11. 使用弹簧剪刀,脊髓在翼芽的水平切开并尾部到肢芽。
    12. 使用镊子,隔离的脊髓被抓住在嘴端 并从胚胎中以单片的形式取出,轻轻地将其拉入   平稳运动(图1g)。
      注意:脊髓是   在该步骤期间保持浸没在PBS中。 在鸡,少量 背脊髓(屋顶板)留下。 孤立 脊髓因此类似于一张床单而不是管。
    13. 将分离的脊髓轻轻地提起到平金属刮刀上 (用于支持)并转移到新的Sylgard衬里的培养皿中 含有4%多聚甲醛的PBS溶液 注意:脊髓是非常 精细,应该轻轻地浸入4%PFA使用封闭 镊子(或另一钝器)。 避免接触脊髓, 如果可能的话。 4%PFA溶液应在室温下,如 如果4%PFA是冷的,脊髓趋于浮动。 打破表面 张力,也可以放置之前解剖的1-2 头部在4%PFA的盘中,在转移脊髓之前。
    14. 平面安装准备(图2)通过仔细钉住生产 脊髓在六个位置(rostrally,medially和caudally on 每侧),使用0.10mm昆虫针(图1h)。内侧表面  脊髓(先前排列的中央管道的封闭  神经管)应朝上。
      注意:将插针倾斜 从脊髓,以防止它们干扰随后的DiI  注射。我们用一个"旗子"(由磁带制成)来标记每本开放书  包裹在0.2mm昆虫针周围),不仅识别胚胎  从中得到了开放的书,但也指示前部 结束准备。
    15. 开放书制剂在4%PFA中在室温下固定30分钟至1小时 注意:打开的书籍不应该被过度修复,因为这会降低DiI扩散的效率并增加背景。
    16. 将固定剂小心地从盘中倒出并用PBS代替 注意:   制剂应冷藏,直到准备注射DiI或 用于显微镜检查。 DiI应在2周内注射。

  2. 用DiI注射开放制剂
    1. 拔针器用于从硼硅酸盐玻璃毛细管制造玻璃微量移液管
    2. 将玻璃微量吸管插入聚乙烯管中
    3. 使用镊子,微量移液管的末端断开,以在尖端获得非常小的直径开口(〜5μm)。
    4. 使用轻轻的吸力将快速DiI(5mg/ml,在乙醇中)吸入微量移液管 注意:  不要画出太多的DiI,以防针尖断裂 在注射过程中,导致DiI溢出和毁了你 样品。将针插入PBS的盘子,轻轻吹出一些。 检查DiI在您之后不会继续从针中漏出 吹完了。如果DiI泄漏,针直径太大。  在这种情况下,请准备新针。
    5. 打开的书准备从下面被照亮,并且识别要注射的区域 注意:  连合神经元的细胞体位于刚刚腹侧 屋顶板和背面的背屈。背部 funiculus可以在开放书中鉴定由于其不同 密度与周围组织相比。 寻找更密集 组织的纵向条纹,位于大约1/5的宽度   从侧面边缘的准备(图1i)。 的 连合神经元直接位于该条带的侧面(即侧面)。   立即背背背屈)。 让你的注射   背侧的侧边缘。
    6. 从一开始 在打开的书的末端,玻璃针插入组织中   角度> 45°,并且当针被抽出时,少量的DiI   轻轻地吹入。
      注意:因为乙醇比PBS密度小, DiI会从注射部位浮出,这可能阻碍你 视图。 尽量不要注入过量的DiI。 你可能需要练习 嘴巴吸取以获得这个权利。
    7. 工作快,DiI 沿着开放的书的长度定期注射 间隔约0.5 mm(图1j) 注意:如果针 变得堵塞,用钳子小心地清除它。 如果尖端变得太大   大(和DiI泄漏),更换针。
    8. 完成每本打开的书的注射后,使用移液管吸走并丢弃任何多余的DiI。
      注意:如果不这样做会导致后台高度。
    9. 注射的开放书准备工作3-4天,以允许DiI沿轴突扩散 注意:如果需要,用PBS补充培养皿,以确保制剂在DiI扩散过程中保持浸泡。

  3. 安装和微观分析
    1. 1ml注射器装有真空油脂并装有18G针
    2. 注射器用于传播薄的,不间断的真空边界 24毫米x 24毫米玻璃盖玻片的边缘周围有油脂。
      注意:   油脂层不宜太厚,否则会很难   在显微镜下可视化制剂。 确保没有 油脂边缘的间隙,或者制剂会变干。 类型   使用的润滑脂也很重要。 低粘度的真空润滑脂可能混合 (在后续步骤中),并产生高背景。
    3. 将几滴无菌PBS加到孔的中间 注意:含甘油与没食子酸正丙酯的固定介质可能与DiI不兼容。
    4. 销钉从开放书中取出,并使用镊子转移到PBS液滴中
    5. 开放的书浸没在PBS中并位于井的中间
    6. 另一个24毫米x 24毫米盖玻片轻轻地放在上面,确保 打开的书保持打开,没有明显的气泡 注意:如有必要,可以移除盖玻片,并重新定位打开的书本。
    7. 对盖玻片的边缘施加轻微的压力以产生完全的油脂密封 注意:在此步骤中,过多的PBS将被挤出。 同样,避免气泡。
    8. 将制备物在黑暗中保存直至准备好 通过荧光显微镜检查和记录(图1k-1)。 将装配的制剂置于玻璃显微镜载玻片的顶部 成像和分析 注意:PBS最终会变干。 分析   应在1-2周内完成,以确保制剂不要   在评估前干燥。


      图1.开放图书准备中的步骤

      1.   HH25-26鸡胚在PBS的皿中。 除去了胚外膜。
      2. 胚胎(去除头部)通过颈部,尾部,翅膀和   四肢。 销钉远离主体成角度。 前方在左边。
      3. 胚胎与心脏和器官删除。 分段的椎骨 现在可见。 用弹簧剪刀剪去的区域创建一个皮瓣 的椎体组织由虚线示出
      4. 一些 椎体在前端已被去除,暴露脊柱 绳索。 在后端,椎骨仍然覆盖脊柱   线。
      5. 去除所有椎骨的暴露脊髓。 DRGs   是可见的。 箭头表示组织的密集线 应该进行纵向切割以将脑膜从中分离 脊髓
      6. 切除脊柱脑膜(箭头)之前和之后的缩略视图
      7. 脊髓从胚胎中拔出后的准备。
      8. 孤立的脊髓,固定在平的"开放书"配置 (参见图2)。 地板躺在中间和背部的   脊髓现在位于横向。 rostrally(R) 内侧(M)和尾侧(C)与准备成角度
      9. 在更高的放大率,组织的致密纵向条纹 对应于背侧曲率由箭头指示。 DiI 应该沿背部的外侧边缘进行注射 字典。
      10. 使用DiI注射部位(红色)安装的开放书准备的明场图像。
      11. 使用DiI注射部位的开放书制备的荧光图像   (红)。 连合轴突,已经生长腹部和交叉 底板,可以看到
      12. 单放大 DiI注射部位。 连合轴突朝向和跨越地板生长 前转。


        图2. 程序。 脊髓解剖出来,在背部开放 屋顶板和放平。 连合神经元(CN,显示为蓝色) 延伸轴突生长腹部和交叉底板(FP)之前 向前转。 连合轴突通过注射DiI标记 (红)。 前,后 ; d背侧; v,腹侧。

笔记

  1. 孤立的脊髓非常脆弱。避免尽可能多地接触它们,并尽量减少/避免溶液的变化。当将脊髓从盘转移到盘时,或当安装DiI注射的开放书用于分析时,总是使用镊子在前端抓住它们。这样,在处理过程中,只有一小部分脊髓会被损坏
  2. 我们以前发表了一个视频协议,演示电穿孔,开放性的解剖和DiI注射步骤,这可能是有用的(Wilson和Stoeckli,2012)。
  3. 有关鸡胚孵化和处理的更多详细信息,请参见参考文献1或参考文献4.
  4. 请参阅通过离体电穿孔(Ito-Ishida,2013)用于口腔移液方法

食谱

  1. 20x无菌PBS(1L)
    160克NaCl
    4克KCl
    28.84g的Na 2 HPO 4 2H 2 O O 将4.14g NaH 2 PO 4钠盐溶液加入其中。
    H O 在高压釜中溶解在1L ddH 2 O中
  2. 4%多聚甲醛(4%PFA)(200ml) 150ml ddH 2 O·dm / 0.8ml 2M NaOH 8克多聚甲醛
    在70℃加热30分钟,冷却,加入20ml 1M PB缓冲液 将pH调节至7.4,用ddH 2 O补足至200ml 储存在4°C
  3. 磷酸盐(PB)缓冲液(1M,1L,pH7.4) 206.6g的Na 2 HPO 4 。 7H O
    加入32g NaH 2 PO 4。 H O 溶解在1L ddH 2 O中

致谢

该协议改编自以前发表的论文Wilson和Stoeckli(2012)和Andermatt等人(2014)。 这项工作得到瑞士国家科学基金会给予E.S.的资助。

参考文献

  1. Andermatt,I.,Wilson,N.and Stoeckli,E.T。(2014)。 电穿孔基于miRNA的质粒以研究基因功能发展中的神经管。 Methods Mol Biol 1101:353-368。
  2. Ito-Ishida,A。(2013)。 通过离体电穿孔在小脑中标记前体颗粒细胞。 生物协议 3(12):e778
  3. Wilson,N.H。和Stoeckli,E.T。(2013)。 Sonic hedgehog以磷脂酰肌醇蛋白聚糖依赖性方式调节其自身受体在后交联合轴突上的表达。 Neuron 79(3):478-491。
  4. Wilson,NH和Stoeckli,ET(2012)。 In ovo
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用:Wilson, N. H. and Stoeckli, E. T. (2014). Open-book Preparations from Chick Embryos and DiI Labeling of Commissural Axons. Bio-protocol 4(13): e1176. DOI: 10.21769/BioProtoc.1176.
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