搜索

Mouse Retinal Whole Mounts and Quantification of Vasculature Protocol
小鼠视网膜整装和脉管系统量化的实验方案   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Angiogenesis is the formation of new blood vessels from a pre-existing vascular bed. It is a multi-step process beginning with enzymatic degradation of the capillary basement membrane, followed by endothelial cell (EC) proliferation, migration, tube formation, assembly of a new basement membrane, and pericyte stabilization. Aberrant angiogenesis plays a major role in the pathogenesis of many diseases. The regulation of this complex process is an important therapeutic target. Success in this pursuit, however, requires the development of in vivo angiogenesis models that provide a reliable and facile platform for mechanistic studies of angiogenic regulation as well as drug development and testing (Carmeliet and Jain, 2011).
Postnatal development of mouse retinal vasculature offers a unique and powerful in vivo angiogenesis model because, unlike other species, mice undergo extensive angiogenesis-dependent maturation of their retinal vessels after birth. As such, this model is also very useful for the mechanistic study of embryonic vascularization (Stahl et al., 2010; Adini et al., 2003).
This protocol describes the steps involved in the whole mount processing of mouse eyes for visualization of the retinal vasculature.

Keywords: Retinal(视网膜), Vessels(容器), Whole mount(整个安装), Mice(老鼠), Angiogenesis(血管生成)

Materials and Reagents

  1. 10% Formalin solution (Sigma-Aldrich, catalog number: HT5011284L ) or Paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15710 )
  2. Triton X-100 (Sigma-Aldrich, catalog number: 93443 )
  3. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  4. Goat serum (Sigma-Aldrich, catalog number: G9023 )
  5. Phosphate buffer saline (PBS) (Sigma-Aldrich, catalog number: P3813 )
  6. Antibody: Bandeiraea simplicifolia BS-1 (LEC)-TRIC (Sigma-Aldrich, catalog number: L-5264 ) diluted 1:200
  7. Mounting medium: ProLong Gold antifade reagents (Life Technologies, catalog number: P36934 )
  8. Blocking buffer (see Recipes)

Equipment

  1. Razor Blade
  2. Shaker-2 min maximum velocity
  3. Epifluorescence microscope fitted with camera
  4. Student Dumont #5 Forceps (Fine Science Tools, catalog number: 9115020 )

  5. Curve Dumont #7 Fine Forceps (Fine Science Tools, catalog number: 1127420 )

  6. Micro dissecting scissors (Fine Science Tools, catalog number: 1501810 )

  7. Microscope cover glasses (Thermo Fisher Scientific, Menzel-Gläser, catalog number: 9161028 )
  8. Microscope glass slides (Thermo Fisher Scientific, Menzel-Gläser, catalog number: 9161145 )
  9. CCD camera (Leica Microsystems, model: DC500 )
  10. Microscope: Nikon Eclipse TE-2000-E fluorescence microscope or Zeiss Inverted Tissue Culture Fluorescence Microscope.

Software

  1. Software Angio Tool (Zudaire et al., 2011) (https://ccrod.cancer.gov/confluence/display/ROB2/Home) or ImageJ software (http://imagej.nih.gov/ij/)

Procedure

  1. In order to visualize vessels and blood flow (leakage of blood as example), perfuse the animal for 10 min by intravenous (IV) injection (200 μl/25 gr of animal) of 20 mg/ml FITC-dextran (2,000 kDa lysine fixable at 20 μg/μl in saline). (Animals should not be anesthetized because anesthetics cause reduced heart rate and blood pressure, requiring longer perfusion times).
  2. Euthanize the animal by using CO2 10 min after IV injection. Enucleate the mouse eye by pulling apart the eyelids, and placing a curved forcep under the eyeball. Close the forcep and grip the connective tissue and optic nerve, being careful to avoid squeezing the eyeball, and gently pull the eyeball from the orbit. Place the eye in 10% Formalin (or 4% PFA) overnight at 4 °C to fix the tissue.
  3. Wash eyes after formalin-fixation 3 times for 15 min with shaking at room temperature in PBS.
  4. After washing the fixed, enucleated eyes, remove the cornea, iris, lens, sclera, and choroid (primary cut around the limbus). Use an 18G needle to make a small hole in the posterior of the limbus. After puncturing the limbus, use micro-scissors to cut around the circumference of the limbus. After finishing the cut, use forceps to remove the cornea, iris, lens, and sclera. Extract the vitreous gel using micro-forceps, or by gently squeezing the. In order to free the retina use two pairs of micro forceps and go around the edges to separate the retina from the eyecup and then gently squeeze the retina out. If necessary, remove the hyaloid vessels with micro forceps (Figure 1).


    Figure 1. Diagram describes the dissection of the eye resulting in a whole mount of the retina

  5. Incubate tissue (retinas) with blocking buffer (materials and reagents) for 1-3 h.
  6. Incubate retinas overnight at 4 °C under gentle agitation in blocking buffer containing BS-1 isolectin (LEC)-TRITC [incubation longer than overnight (12-24 h) may increase nonspecific fluorescence].
  7. Wash retinas 4-5 times (1 h between washes) in PBS at room temperature on a shaker.
  8. In order to flatten and mount the retina under a coverslip, make four incisions as shown in Figure 2.


    Figure 2. Retina cup is cut with surgical blade in order to allow the retinas to lie flat on the slide

  9. Place the retina onto a slide with the eyecup facing up, and cut the retina in each quarter with a micro-scissor. Using a wide-bore plastic Pasteur pipette, remove excess PBS and spread the eyecup open like a flower. Add 1 drop (around 10 μl) of mounting media over the retina and cover with a coverslip.
  10. Images of flat-mounted retinal vascular plexus are acquired with an epifluorescence microscope.
    To capture 3D images of the retinal vascular plexus, use a confocal microscope fitted with a CCD camera. A 5x or 10x objective offers the best image resolution for Angio Tool software.
  11. Angio Tool image software can be used for quantifying numerous features of the angiogenic vessels including vascular density, number of branch points and tip cells, and others (Zudaire et al., 2011).

Representative data



Figure 3. Representative whole mounts retina.
Retinas were dissected and labeled (Alexa Fluor 594-conjugated isolectin). Scale bar: 1 mm (<5% the quality of the fluorescence-labeled are poor).

Notes

Thoroughly washing the retinas after labeling with BS-1 (LEC)-TRTIC is critical, do not reduce the washing step.

Recipes

  1. Blocking buffer
    PBS containing 0.5% triton X-100, 0.02% sodium azide, 10% goat serum

Acknowledgments

This work was adapted and modified from previous work supported by US Public Health Service NIH grant HL071049 and part by a grant from the NIH (R01EY012726).

References

  1. Adini, I., Rabinovitz, I., Sun, J. F., Prendergast, G. C. and Benjamin, L. E. (2003). RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. Genes Dev 17(21): 2721-2732.
  2. Carmeliet, P. and Jain, R. K. (2011). Molecular mechanisms and clinical applications of angiogenesis. Nature 473(7347): 298-307.
  3. Stahl, A., Connor, K. M., Sapieha, P., Chen, J., Dennison, R. J., Krah, N. M., Seaward, M. R., Willett, K. L., Aderman, C. M. and Guerin, K. I. (2010). The mouse retina as an angiogenesis model. Invest Ophth Vis Sci 51(6): 2813-2826.
  4. Zudaire, E., Gambardella, L., Kurcz, C. and Vermeren, S. (2011). A computational tool for quantitative analysis of vascular networks. PLoS One 6(11): e27385.

简介

血管生成是从预先存在的血管床形成新的血管。它是一个多步骤的过程,从毛细血管基底膜的酶降解开始,然后是内皮细胞(EC)增殖,迁移,管形成,新基底膜的装配和周细胞稳定。异常血管生成在许多疾病的发病机理中起重要作用。这种复杂过程的调节是重要的治疗靶标。然而,这种追求的成功需要开发在体内血管生成模型,其为血管生成调节以及药物开发和测试的机械研究提供可靠和容易的平台(Carmeliet和Jain,2011)。
小鼠视网膜血管系统的产后发育提供了一种独特且强大的体内血管生成模型,因为与其他物种不同,小鼠在出生后经历其视网膜血管的广泛的血管生成依赖性成熟。因此,该模型对于胚胎血管形成的机械研究也是非常有用的(Stahl等人,2010; Adini等人,2003)。
此协议描述了整个安装处理鼠标眼睛以便视网膜血管系统可视化的步骤。

关键字:视网膜, 容器, 整个安装, 老鼠, 血管生成

材料和试剂

  1. 10%福尔马林溶液(Sigma-Aldrich,目录号:HT5011284L)或多聚甲醛(PFA)(Electron Microscopy Sciences,目录号:15710)
  2. Triton X-100(Sigma-Aldrich,目录号:93443)
  3. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  4. 山羊血清(Sigma-Aldrich,目录号:G9023)
  5. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P3813)
  6. 抗体:以1:200稀释的Bandeiraea simplicifolia BS-1(LEC)-TRIC(Sigma-Aldrich,目录号:L-5264)
  7. 安装介质:ProLong Gold防褪色试剂(Life Technologies,目录号:P36934)
  8. 阻止缓冲区(参见配方)

设备

  1. 剃刀刀片
  2. 振动器-2分钟最大速度
  3. 带有相机的荧光显微镜
  4. 学生Dumont#5镊子(Fine Science Tools,目录号:9115020)

  5. Curve Dumont#7 Fine Forceps(Fine Science Tools,目录号:1127420)

  6. 微解剖剪刀(Fine Science Tools,目录号:1501810)

  7. 显微镜盖玻璃(Thermo Fisher Scientific,Menzel-Gläser,目录号:9161028)
  8. 显微镜载玻片(Thermo Fisher Scientific,Menzel-Glüser,目录号:9161145)
  9. CCD照相机(Leica Microsystems,型号:DC500)
  10. 显微镜:Nikon Eclipse TE-2000-E荧光显微镜或Zeiss Inverted Tissue Culture荧光显微镜。

软件

  1. 软件Angio工具(Zudaire ,,2011)( https: //ccrod.cancer.gov/confluence/display/ROB2/Home )或ImageJ软件( http://imagej.nih.gov/ij/

程序

  1. 为了可视化血管和血流(作为实例的血液泄漏),通过静脉内(IV)注射(200
    图1.图描述了眼睛的解剖,导致整个视网膜

  2. 用封闭缓冲液(材料和试剂)孵育组织(视网膜)1-3小时。
  3. 孵育视网膜过夜在温和搅拌下在含有BS-1同工凝集素(LEC)-TRITC [孵育时间过夜(12-24小时)的封闭缓冲液可能增加非特异性荧光]。
  4. 在室温下在振动器上在PBS中洗涤视网膜4-5次(洗涤之间1小时)
  5. 为了使视网膜平整和安装在盖玻片下,做四个切口,如图2所示。


    图2.视网膜杯用手术刀片切割,以允许视网膜平放在幻灯片上

  6. 将视网膜放在幻灯片上,眼罩朝上,用微型剪刀切割每个季度的视网膜。使用广口的塑料巴斯德吸管,删除多余的PBS和传播的眼罩开放像一朵花。在视网膜上添加1滴(大约10个μ l)的封装介质,并盖上盖玻片。
  7. 使用落射荧光显微镜获得平面安装的视网膜血管丛的图像。
    要捕获视网膜血管丛的3D图像,请使用配有CCD相机的共聚焦显微镜。 5x或10x目标为Angio Tool软件提供最佳的图像分辨率。
  8. Angio Tool图像软件可用于量化血管生成血管的许多特征,包括血管密度,分支点和尖端细胞的数目等(Zudaire等人,2011)。

代表数据



图3.代表整个视网膜。 解剖视网膜并标记(Alexa Fluor 594-共轭的异凝集素)。 比例尺:1mm(<5%,荧光标记的质量较差)。

笔记

用BS-1(LEC)-TRTIC标记后彻底清洗视网膜是关键,不要减少洗涤步骤。

食谱

  1. 阻塞缓冲区
    含有0.5%triton X-100,0.02%叠氮化钠,10%山羊血清的PBS

致谢

这项工作改编和修改以前的工作由美国公共卫生服务NIH授予HL071049支持和部分由NIH(R01EY012726)授权。

参考文献

  1. Adini,I.,Rabinovitz,I.,Sun,J.F.,Prendergast,G.C。和Benjamin,L.E。(2003)。 RhoB控制血管发育过程中Akt运输和内皮细胞的阶段特异性存活。 em> Genes Dev 17(21):2721-2732。
  2. Carmeliet,P.和Jain,R.K。(2011)。 血管生成的分子机制和临床应用 Nature 473 (7347):298-307。
  3. Stahl,A.,Connor,K.M.,Sapieha,P.,Chen,J.,Dennison,R.J.,Krah,N.M.,Seaward,M.R.,Willett,K.L.,Aderman,C.M.and Guerin,K.I。 小鼠视网膜作为血管生成模型。 Invest Ophth Vis Sci 51(6):2813-2826。
  4. Zudaire,E.,Gambardella,L.,Kurcz,C.and Vermeren,S。(2011)。 血管网络定量分析的计算工具 PLoS One < em> 6(11):e27385。
  • 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。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Adini, I. and Ghosh, K. (2015). Mouse Retinal Whole Mounts and Quantification of Vasculature Protocol. Bio-protocol 5(15): e1546. DOI: 10.21769/BioProtoc.1546.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。