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Embryonic Intravitreous Injection in Mouse

小鼠胚胎期玻璃体腔注射

LC Liyuan Cui
YD Yupu Diao
JZ Jiayi Zhang

发布: 2018年07月20日第8卷第14期 DOI: 10.21769/BioProtoc.2929 浏览次数: 6697

评审: Miao HeEhsan KheradpezhouhAnonymous reviewer(s)

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Abstract

Axons of retinal ganglion cells (RGCs) relay visual information from the retina to lateral geniculate nucleus (LGN) and superior colliculus (SC), which are two major image-forming visual nuclei. Wiring of these retinal projections completes before vision begins. However, there are few studies on retinal axons at embryonic stage due to technical difficulty. We developed a method of embryonic intravitreous injection of dyes in mice to visualize retinal projections to LGN and SC. This study opens up the possibility of understanding early visual circuit wiring in mice embryos.

Keywords: Embryo (胚胎) search Intravitreous injection (玻璃体腔注射) search Retinal projection (视网膜投影) search Retinal axons (视网膜轴突) search Uterus (子宫) search

Background

Retinal axons begin to project to LGN and SC as early as embryonic day 14.5 (E14.5) in mice. To investigate axon projections in embryos, dyes need to be injected intravitreally while keeping the embryos alive for at least 10 h prior to injection surgery. Previous studies showed embryos can be cultured in vitro using mouse serum with oxygen. However, issues such as nutrition diversity, oxygen saturation and temperature regulation impinge the physiological condition of the embryo. In our research, we conducted intravitreous injection through uterine wall and eyelids in embryos and kept them in uterine after injection. Retinal axon projections to LGN and SC were nicely labeled from E15.5 to E18.5.

Materials and Reagents

  1. 50 ml centrifuge tube (Corning, catalog number: 430828 )
  2. Cotton ball (Winner Medical Group, catalog number: 50401050 )
  3. Sterile gauze (Winner Medical Group, catalog number: 016935 )
  4. Suture needle (Ningbo Medical Needle Co., LTD, 7/0)
  5. Microscope slides (Fisher Scientific, catalog number: 12-550-15 )
  6. Microscope cover glass (Fisher Scientific, catalog number: 12-544-18 )
  7. Dropper (Shanghai Baiqian Biotechnology, catalog number: J00082 )
  8. Mouse (Shanghai SLAC, strain: C57BL/6J)
  9. Redistilled water (ddH2O)
  10. 75% alcohol (Sinopharm Chemical Reagent, catalog number: 80176960 )
  11. Mineral oil (Sigma-Aldrich, catalog number: M8410 )
  12. Cholera toxin B (CTB), Alexa FluorTM 555 conjugate (Thermo Fisher Scientific, InvitrogenTM, catalog number: C22843 )
  13. Isoflurane (RWD Life Science, catalog number: R510-22 )
  14. Iodophor (Shanghai Likang Disinfectant Hi-Tech, catalog number: 310100 )
  15. Lidocaine (MP Biomedicals, catalog number: 190111 )
  16. Sucrose (AMRESCO, catalog number: M117 )
  17. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 16005 )
  18. Optimal cutting temperature compound (O.C.T) (Sakura, Tissue-Tek®, catalog number: 4583 )
  19. Diamidino-phenyl-indole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  20. AQUA-MountTM Mounting medium (Thermo Fisher Scientific, catalog number: 13800 )
  21. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  22. Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Sigma-Aldrich, Fluka, catalog number: 71645 )
  23. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 )
  24. Ampicillin (INALCO, catalog number: 1758-9314 )
  25. Trichloroacetaldehyde hydrate (Sinopharm Chemical Reagent, catalog number: 30037517 )
  26. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S5011 )
  27. Tris (Sangon Biotech, catalog number: A100826 )
  28. Tris hydrochloride (Tris-HCl) (AMRESCO, catalog number: T0234 )
  29. Triton X-100 (AMRESCO, catalog number: 0694 )
  30. DAPI (Sigma-Aldrich, catalog number: D9542 )
  31. 0.1 M Phosphate buffer saline solution(PBS) (see Recipes)
  32. Ampicillin solution (see Recipes)
  33. 0.9% Sodium chloride solution (see Recipes)
  34. 1% lidocaine solution (see Recipes)
  35. 10% Chloral hydrate solution (see Recipes)
  36. 0.2 M Phosphate buffer (PB) (see Recipes)
  37. 0.05 M Tris buffered saline (TBS) (see Recipes)
  38. 0.5%/0.05% Triton solution (see Recipes)
  39. CTB solution (see Recipes)
  40. 4% paraformaldehyde solution (see Recipes)
  41. 30% sucrose solution (see Recipes)
  42. DAPI solution (see Recipes)

Equipment

  1. Glass Capillaries for Nanoliter 2010 (referred to as pipette in this manuscript) (World Precision Instruments, catalog number: 504949 )
  2. Scissors (RWD Life Science, catalog number: S12003-09 )
  3. Tweezers (VETUS, catalog number: ST-10 )
  4. Ophthalmic scissors (World Precision Instruments, catalog number: 14003-G )
  5. Water bath (Jinghong Experimental Equipment, model: XMID-8222 )
  6. Nanoject II Auto-Nanoliter Injector (Drummond Scientific, model: Nanoject II , catalog number: 6584)
  7. DC temperature controller (FHC, model: 40-90-8D )
  8. Anesthesia machine (RWD Life Science, catalog number: R610 )
  9. Flaming/brown micropipette puller (Sutter Instrument, model: P-97 )
  10. Shaver (Codos, catalog number: KP-3000 )
  11. Fluorescence microscope (Nikon Instruments, model: Eclipse Ni-U )
  12. -80 °C freezer (Thermo Fisher Scientific, catalog number: ULT 1386-3-V42 )
  13. Freezing microtome (Leica Biosystems, model: Leica CM1950 )

Software

  1. ImageJ (NIH, USA)
  2. Matlab (Mathworks Inc, USA)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Cui, L., Diao, Y. and Zhang, J. (2018). Embryonic Intravitreous Injection in Mouse. Bio-protocol 8(14): e2929. DOI: 10.21769/BioProtoc.2929.

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分类

神经科学 > 神经解剖学和神经环路 > 视神经

神经科学 > 感觉和运动系统 > 视觉系统

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