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
发布: 2014年07月05日第4卷第13期 DOI: 10.21769/BioProtoc.1176 浏览次数: 12623
评审: Xuecai Ge
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参见作者原研究论文
The authors used this protocol in:
Aug 2013
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
- Fertilized eggs containing chicken embryos (incubated until Hamburger and Hamilton stage 25-26)
- PBS (add 50 ml of 20x PBS to 950 ml of ddH2O)
- Sylgard silicone elastomer (World Precision Instruments, catalog number: SYLG184 )
- Fast DiI (Life Technologies, Molecular Probes®, catalog number: D-7756 ) (dissolved at 5 mg/ml in ethanol)
- Vacuum grease (Dow Corning Corporation, catalog number: 976V )
- 20x sterile phosphate buffered saline (PBS) (see Recipes)
- 4% paraformaldehyde (4% PFA) (see Recipes)
- Phosphate (PB) buffer (1 M, 1 L, pH 7.4) (see Recipes)
Equipment
- Incubator set at 38.5 °C and 45% humidity (e.g. FIEM, Juppiter 576 Setter+Hatcher; Kendro Laboratory Products, Heraeus/Kendro, model: B12 )
- Spring scissors (Fine Science Tools, catalog number: 15003-08 )
- Dumont #5 forceps (Fine Science Tools, catalog number: 11252-20 )
- 90 mm Petri dishes (e.g. Thermo Fisher Scientific, Sterilin®, catalog number: 101VR20/C )
- 0.20 mm insect pins (Fine Science Tools, catalog number: 26002-20 )
- 0.10 mm insect pins (Fine Science Tools, catalog number: 26002-10 )
- Microscope with adjustable transmitted light source (Leica MZ6 stereomicroscope with Leica CLS 150x )
- Fine surgical scalpel (Grieshaber Logistic Group, catalog number: 68101 )
- Tungsten wire (0.075 mm) (World Precision Instruments, catalog number: TGW0325 )
- Metal flat spatula/spoon (e.g. Sigma-Aldrich, catalog number: S4022 , S3397 )
- Borosilicate glass capillaries (outer Ø/inner Ø: 1.2 mm/0.68 mm) (World Precision Instruments, catalog number: 1B120F-4 )
- Glass needle puller (Narishige Group, catalog number: PC-10 )
- Polyethylene tubing (Ø 1.24 mm) (e.g. Angst and Pfister AG, catalog number: FT0110350018 )
- Plastic transfer pipettes (e.g. Sarstedt, catalog number: 86.1171 )
- 18 G x 1.5” needle (e.g. Braun, catalog number: 4665120 )
- 1 ml syringe (e.g. Braun, catalog number: 9166017V )
- 20 ml syringe (e.g. Braun, catalog number : 4606205V )
- 24 mm x 24 mm glass coverslips (VWR International, catalog number: 631-0127 )
- Microscope suitable for brightfield and fluorescent microscopy (e.g. Olympus, model: BX51 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
神经科学 > 发育 > 神经元
神经科学 > 神经解剖学和神经环路 > 动物模型
发育生物学 > 细胞生长和命运决定 > 神经元
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