参见作者原研究论文

本实验方案简略版
Mar 2017
Advertisement

本文章节


 

Embryonic Intravitreous Injection in Mouse
小鼠胚胎期玻璃体腔注射   

引用 收藏 提问与回复 分享您的反馈 Cited by

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), Intravitreous injection (玻璃体腔注射), Retinal projection (视网膜投影), Retinal axons (视网膜轴突), Uterus (子宫)

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

  1. Put one male mouse and two female mice into a home cage at 20:00. Detect the presence of Vaginal plug at 8:00 AM next day. If the female mouse has a vaginal plug, the embryonic stage is denoted by 0.5 days (E0.5). Otherwise, divide male and female mice into two home cages and put them into one home cage again at 20:00.
  2. Sterilize all metal surgical instruments for 30 min at a station of high temperature (121 °C) and high pressure (0.12 MPa). Disinfect the other nonmetal apparatuses with 75% ethanol.
  3. Fill PBS and ampicillin solution into two centrifuge tubes respectively. Keep the tubes in a water bath at 45 °C.
  4. Pull a Pipette using a micropipette puller. Break the Pipette tip with scissors to make it open. Fill the Pipette with mineral oil and then fit it in Nanoject II. CTB-555 is inhaled into pipette using Nanoject II.
    Note: The diameter of the pipette tip is about 50 μm.
  5. Anesthetize the pregnant mouse with 1.5% isoflurane in oxygen in a chamber until its respiratory frequency decreases to 60 times per min and then transfer it onto a heat pad at 32 °C controlled by DC temperature controller.
  6. Place the head of the pregnant mouse into a face mask to make sure that mouse is anesthetized by 0.5-1% isoflurane in oxygen continuously until respiratory frequency maintains between 80-100 times per min.
  7. Shave the mouse abdomen and disinfect successively by using a cotton ball with 75% ethanol and iodophor.
  8. Cover the mouse abdomen with a sterile gauze which is cut a 2-cm long slit to expose the position of the abdominal incision.
  9. Slit the abdominal skin and peritoneum carefully at the middle of abdomen from outside to inside.
    Note: Incision at the precise middle of abdomen can avoid capillary vessels. Otherwise, bleeding induced by damaged capillary vessels is easier to cause wound infection.
  10. Pull Embryos out carefully with tweezers and place them on dry sterile gauze over the abdomen.
    Note: The tension should be slight to avoid tearing uterine wall. Be careful not to damage vessels and placenta.
  11. Cut a slit (~1 mm) on the uterine wall over the eyeball of the embryo a slit (~1 mm) using ophthalmic scissors (Video 1).
    Note: If there are vessels on the uterine wall over the eyeball of the embryo, the embryo is rotated by hand softly to avoid vessels.

    Video 1. The posture of intravitreous injection

  12. One hand holds embryo. Another hand holds Nanoject II. Pipette pass through the slit of uterine wall, eye slide, sclera and then plug it in vitreous chamber. Inject 1 μl CTB-555 into the vitreous chamber of one eye at the speed of 69 nl per time, 1-2 sec interval (Video 1).
    Notes:
    1. One millimeter long pipette tip is inserted subretinally. After injection, the pipette stays in the eye for 5 sec.
    2. A second person adds warm PBS using a dropper to the surface of uterine wall to keep it moist throughout the whole process.
    3. Resting the arms of the experimentalist on the table helps to prevent disturbance during the injection process.
  13. Inject eyes of other embryos successively with the similar volume and speed.
  14. Replace the embryos into abdomen carefully with tweezers.
  15. Fill the abdomen cavity of the pregnant mouse with 1 ml ampicillin solution to prevent infection. Absorb the extra solution with gauze over the abdomen.
  16. Suture the peritoneum and abdominal skin from inside to outside.
  17. Apply 1% lidocaine solution on the wound to relieve the pain.
  18. Anesthetize the pregnant mouse continually with 0.5-1% isoflurane for 10 h.
    Note: The pregnant mouse is anesthetized to make the surgical procedure smooth.
  19. After 10 h, anesthetize pregnant mouse with 10% chloral hydrate solution at the dose of 0.06 ml per 10 g weight.
    Note: The pregnant mouse still has a heartbeat after chloral hydrate solution injection to avoid fetal hypoxia.
  20. Cut the abdomen open again to expose embryos. Made a slit on the uterine wall using scissors. Cut the umbilical cord to take out embryo.
  21. Fix the embryo on a board and implement heart perfusion using 4% PFA solution.
    Note: The process of transcardial perfusion of embryos is similar to that of adults. It is best to perform transcardial perfusion under an atomic microscope to reveal the structure of the heart.
  22. Take brains of embryos out and fix in 4% PFA for 12-18 h.
    Note: Retinas of embryos are taken out and fixed in 4% PFA for 2 h. Retinas are mounted on slides and observed with the fluorescence microscope. It is necessary to make sure that majority of retinal ganglion cells are labeled by CTB-555.
  23. Dehydrate brains in 30% sucrose.
  24. Embed brains in O.C.T after they sink in 30% sucrose solution, and then rapidly transfer into a -80 °C freezer. Half an hour later, take the brains into freezing microtome to rewarm for 20 min.
  25. Cut brains into coronal slices of 20 μm thick. Wash the brain slices 5 times (5 min per time) using TBS solution to remove O.C.T away.
    Note: All brain slices of LGN and SC section are collected.
  26. Stain brain slices with DAPI (see Recipe 12) for 7 min, and then wash 5 times with TBS solution (how long each time).
  27. Mount brain slices on microscope slides. Apply mounting medium on slices until slices are dry in the air without light. Finally, coverslips are mounted on slices.
  28. Take pictures of brain slices under a fluorescence microscope (Figure 1). The results of embryonic intravitreous injection can also be found online at: https://academic.oup.com/cercor/article/28/4/1168/3064954.


    Figure 1. Images of LGN labeled with CTB-555 after embryonic intravitreous injection at E18.5. The dorsal LGN is outlined with white dashed lines.

Data analysis

Analysis of retinal projection was conducted as previously described (from our lab) (Diao et al., 2018). Briefly, the background of all images was subtracted in ImageJ (NIH, USA). Quantifications of fluorescently labeled areas, such as the fraction of contralateral projections and sizes of LGN and SC, were performed using Matlab (Mathworks Inc, USA). For 20 μm brain slices, a threshold of 30% (fluorescence intensity of pixels above 30% of the maximum intensity) is applied in the calculation of fraction of contralateral projections. CTB-555 and DAPI fluorescent signals helped outlining the territory of LGN and SC.
Note: The birth-dating of embryos could be differed by ± 0.5 days. It is necessary to use sufficient number of pregnant females to obtain sufficient embryos for analysis.

Recipes

  1. 0.1 M Phosphate buffer saline solution (PBS)
    0.02 M KH2PO4, 0.04 M Na2HPO4·2H2O and 0.1567 M NaCl in ddH2O
  2. Ampicillin solution
    0.1 mg/ml ampicillin in 0.1 M PBS
  3. 0.9% Sodium chloride solution
    0.9% NaCl in ddH2O
  4. 1% lidocaine solution
    1% lidocaine in 0.9% NaCl solution
    Note: Add a little of hydrochloric acid into the solution to help dissolve.
  5. 10% Chloral hydrate solution
    10% Chloral hydrate in 0.9% NaCl solution
  6. 0.2 M Phosphate buffer (PB)
    0.04 M NaH2PO4 and 0.2 M Na2HPO4·2H2O in ddH2O
  7. 0.05 M Tris buffered saline (TBS)
    0.0123 M Tris, 0.0377 M Tris-HCl, 0.1538 M NaCl in ddH2O
  8. 0.5%/0.05% Triton solution
    0.5%/0.05% Triton X-100 in TBS
  9. CTB solution
    500 μg CTB solid powder is dissolved by 10 μl dimethyl sulphoxide (DMSO) and then add 230 μl 0.9% sodium chloride solution
  10. 4% paraformaldehyde solution
    4% PFA in PB
  11. 30% sucrose solution
    30% sucrose in PB
  12. DAPI solution
    1 mg/ml DAPI in ddH2O for storage
    1:3,000 diluted in TBS for working concentration

Acknowledgments

The authors declare no competing interests. This protocol was adapted from Diao et al. (2018). We thank the following funding agencies for supporting this work: the NSF of China (31421091 and 31422025), the Young 1000 Plan and Ministry of Science and Technology of the People’s Republic of China (2015AA020512).

References

  1. Diao, Y., Cui, L., Chen, Y., Burbridge, T. J., Han, W., Wirth, B., Sestan, N., Crair, M. C. and Zhang, J. (2018). Reciprocal connections between cortex and thalamus contribute to retinal axon targeting to dorsal lateral geniculate nucleus. Cereb Cortex 28(4): 1168-1182.

简介

视网膜神经节细胞(RGC)的轴突将视觉信息从视网膜传递到外侧膝状核(LGN)和上丘(SC),这是两个主要的成像视觉核。 在视力开始之前完成这些视网膜投射的布线。 然而,由于技术困难,很少有关于胚胎期视网膜轴突的研究。 我们开发了一种胚胎玻璃体内注射染料的方法,用于观察LGN和SC的视网膜投射。 这项研究开辟了理解小鼠胚胎早期视觉电路布线的可能性。

【背景】早在胚胎第14.5天(E14.5)小鼠中,视网膜轴突开始向LGN和SC投射。 为了研究胚胎中的轴突投射,需要在注射手术前保持胚胎存活至少10小时,玻璃内注射染料。 以前的研究表明,胚胎可以使用含氧小鼠血清进行体外培养。 然而,营养多样性,氧饱和度和温度调节等问题会影响胚胎的生理状况。 在我们的研究中,我们通过胚胎中的子宫壁和眼睑进行玻璃体内注射,并在注射后将它们保存在子宫中。 从E15.5到E18.5很好地标记了对LGN和SC的视网膜轴突投射。

关键字:胚胎, 玻璃体腔注射, 视网膜投影, 视网膜轴突, 子宫

材料和试剂

  1. 50毫升离心管(康宁,目录号:430828)
  2. 棉球(Winner Medical Group,目录号:50401050)
  3. 无菌纱布(Winner Medical Group,目录号:016935)
  4. 缝合针(宁波医用针有限公司,7/0)
  5. 显微镜载玻片(Fisher Scientific,目录号:12-550-15)
  6. 显微镜盖玻片(Fisher Scientific,目录号:12-544-18)
  7. 滴管(上海百千生物技术,目录号:J00082)
  8. 小鼠(上海SLAC,菌株:C57BL / 6J)
  9. 重蒸水(ddH 2 O)
  10. 75%酒精(国药化学试剂,目录号:80176960)
  11. 矿物油(Sigma-Aldrich,目录号:M8410)
  12. 霍乱毒素B(CTB),Alexa Fluor TM 555结合物(Thermo Fisher Scientific,Invitrogen TM ,目录号:C22843)
  13. 异氟烷(RWD生命科学,目录号:R510-22)
  14. Iodophor(上海利康消毒高科技,目录号:310100)
  15. 利多卡因(MP Biomedicals,目录号:190111)
  16. 蔗糖(AMRESCO,目录号:M117)
  17. 多聚甲醛(PFA)(西格玛奥德里奇,目录号:16005)
  18. 最佳切削温度化合物(O.C.T)(Sakura,Tissue-Tek ®,目录号:4583)
  19. 二脒基 - 苯基 - 吲哚(DAPI)(Sigma-Aldrich,目录号:D9542)
  20. AQUA-Mount TM 安装介质(赛默飞世尔科技,目录号:13800)
  21. 磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:P5655)
  22. 磷酸氢二钠二水合物(Na 2 HPO 4 ·2H 2 O)(Sigma-Aldrich,Fluka,目录号:71645)
  23. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S5886)
  24. 氨苄西林(INALCO,目录号:1758-9314)
  25. 三氯乙醛水合物(国药化学试剂,目录号:30037517)
  26. 磷酸二氢钠(NaH 2 PO 4 )(Sigma-Aldrich,目录号:S5011)
  27. Tris(Sangon Biotech,目录号:A100826)
  28. 盐酸三(Tris-HCl)(AMRESCO,目录号:T0234)
  29. Triton X-100(AMRESCO,目录号:0694)
  30. DAPI(Sigma-Aldrich,目录号:D9542)
  31. 0.1 M磷酸盐缓冲盐水溶液(PBS)(见食谱)
  32. 氨苄青霉素溶液(见食谱)
  33. 0.9%氯化钠溶液(见食谱)
  34. 1%利多卡因溶液(见食谱)
  35. 10%水合氯醛溶液(见食谱)
  36. 0.2 M磷酸盐缓冲液(PB)(见食谱)
  37. 0.05M Tris缓冲盐水(TBS)(见食谱)
  38. 0.5%/ 0.05%Triton溶液(见食谱)
  39. CTB解决方案(见食谱)
  40. 4%多聚甲醛溶液(见食谱)
  41. 30%蔗糖溶液(见食谱)
  42. DAPI解决方案(见食谱)

设备

  1. Nanoliter 2010玻璃毛细管(本手稿中称为移液器)(World Precision Instruments,目录号:504949)
  2. 剪刀(RWD生命科学,目录号:S12003-09)
  3. 镊子(VETUS,目录号:ST-10)
  4. 眼科剪刀(World Precision Instruments,目录号:14003-G)
  5. 水浴(景洪实验设备,型号:XMID-8222)
  6. Nanoject II Auto-Nanoliter Injector(Drummond Scientific,型号:Nanoject II,目录号:6584)
  7. 直流温度控制器(FHC,型号:40-90-8D)
  8. 麻醉机(RWD生命科学,目录号:R610)
  9. 火焰/棕色微量移液器拉拔器(Sutter Instrument,型号:P-97)
  10. 剃须刀(Codos,目录号:KP-3000)
  11. 荧光显微镜(Nikon Instruments,型号:Eclipse Ni-U)
  12. -80°C冰箱(Thermo Fisher Scientific,目录号:ULT 1386-3-V42)
  13. 冷冻切片机(Leica Biosystems,型号:Leica CM1950)

软件

  1. ImageJ(美国国立卫生研究院)
  2. Matlab(Mathworks公司,美国)

程序

  1. 在20:00将一只雄性小鼠和两只雌性小鼠放入家笼中。第二天上午8点检测阴道塞的存在。如果雌性小鼠具有阴道栓,则胚胎期表示0.5天(E0.5)。否则,将雄性和雌性小鼠分成两个家笼,并在20:00再次将它们放入一个家笼中。
  2. 在高温(121°C)和高压(0.12 MPa)的站点对所有金属手术器械进行灭菌30分钟。用75%乙醇消毒其他非金属设备。
  3. 将PBS和氨苄青霉素溶液分别装入两个离心管中。将管保持在45°C的水浴中。
  4. 使用微量移液器拉出器拉动移液器。用剪刀打破移液器吸头使其打开。用移液管填充矿物油,然后将其装入Nanoject II中。使用Nanoject II将CTB-555吸入移液管中。
    注意:移液器吸头的直径约为50μm。
  5. 在室内用氧气中的1.5%异氟烷麻醉怀孕的小鼠,直到其呼吸频率降低到每分钟60次,然后将其转移到32°C的加热垫上,由DC温度控制器控制。
  6. 将怀孕老鼠的头部放入面罩中,确保小鼠连续用氧气中的0.5-1%异氟烷麻醉,直至呼吸频率保持在每分钟80-100次之间。
  7. 刮除小鼠腹部并使用含75%乙醇和碘伏的棉球连续消毒。
  8. 用无菌纱布盖住小鼠腹部,切开2厘米长的切口以露出腹部切口的位置。
  9. 从腹部到腹部,从腹部中间小心地切开腹部皮肤和腹膜。
    注意:腹部精确中部的切口可以避免毛细血管。否则,受损的毛细血管引起的出血更容易引起伤口感染。
  10. 用镊子小心地拉出胚胎,然后将它们放在腹部干燥的无菌纱布上。
    注意:张力应轻微,以免撕裂子宫壁。小心不要损坏血管和胎盘。
  11. 使用眼科剪刀(视频1)在胚胎眼球上方的子宫壁上切开一条狭缝(~1 mm)(约1 mm)。
    注意:如果胚胎眼球上的子宫壁上有血管,用手轻轻旋转胚胎以避免血管。

    视频1
  12. 一只手拿着胚胎。另一只手拿着Nanoject II。移液器通过子宫壁,眼睛滑动,巩膜切开,然后将其塞入玻璃体腔。将1μlCTB-555注射到一只眼睛的玻璃体腔内,每次69 nl,间隔1-2秒(视频1)。
    注意:
    1. 一个毫米长的移液管尖端在视网膜下插入。注射后,移液器在眼睛中停留5秒钟。
    2. 第二个人使用滴管将温热的PBS添加到子宫壁表面,以使其在整个过程中保持湿润。
    3. 将实验者的手臂放在桌子上有助于防止注射过程中的干扰。
  13. 以相似的体积和速度连续注射其他胚胎的眼睛。
  14. 用镊子小心地将胚胎换成腹部。
  15. 用1ml氨苄青霉素溶液填充怀孕小鼠的腹腔,以防止感染。在腹部用纱布吸收额外的溶液。
  16. 从内到外缝合腹膜和腹部皮肤。
  17. 在伤口上涂抹1%利多卡因溶液以缓解疼痛。
  18. 用0.5-1%异氟醚连续麻醉孕鼠10小时。
    注意:怀孕的老鼠被麻醉,使手术过程顺利。
  19. 10小时后,用10%水合氯醛溶液麻醉怀孕小鼠,剂量为0.06ml / 10g体重。
    注意:注射水合氯醛溶液后怀孕的小鼠仍有心跳,以避免胎儿缺氧。
  20. 再次打开腹部露出胚胎。用剪刀在子宫壁上切开。切断脐带取出胚胎。
  21. 将胚胎固定在板上并使用4%PFA溶液进行心脏灌注。
    注意:经心脏灌注胚胎的过程与成人相似。最好在原子显微镜下进行心脏灌注以显示心脏的结构。
  22. 取出胚胎大脑,用4%PFA固定12-18小时。
    注意:取出胚胎的视网膜并在4%PFA中固定2小时。将视网膜安装在载玻片上并用荧光显微镜观察。有必要确保大多数视网膜神经节细胞都被CTB-555标记。
  23. 用30%蔗糖脱水大脑。
  24. 将它们沉入30%蔗糖溶液中后,将其嵌入O.C.T中,然后迅速转移到-80°C冰箱中。半小时后,将大脑放入冷冻切片机中,恢复20分钟。
  25. 将大脑切成20微米厚的冠状切片。使用TBS溶液清洗脑切片5次(每次5分钟)以除去O.C.T.
    注意:收集LGN和SC部分的所有脑片。
  26. 用DAPI染色脑切片(见配方12)7分钟,然后用TBS溶液洗涤5次(每次多长时间)。
  27. 在显微镜幻灯片上装载脑切片。将切片介质涂在切片上,直到切片在空气中干燥,没有光照。最后,盖玻片安装在切片上。
  28. 在荧光显微镜下拍摄脑切片(图1)。胚胎玻璃体腔注射的结果也可以在网上找到:
    https://academic.oup.com/cercor/article/28/4 / 1168/3064954 。


    图1.在E18.5胚胎玻璃体内注射后用CTB-555标记的LGN图像。背侧LGN用白色虚线勾勒出来。

数据分析

视网膜投射的分析如前所述(来自我们的实验室)进行(Diao et al。,2018)。简而言之,在ImageJ(NIH,USA)中减去所有图像的背景。使用Matlab(Mathworks Inc,USA)进行荧光标记区域的定量,例如对侧投影的分数和LGN和SC的大小。对于20μm脑切片,在计算对侧投影的分数时应用30%的阈值(像素的荧光强度高于最大强度的30%)。 CTB-555和DAPI荧光信号有助于概述LGN和SC的领土。
注意:胚胎的出生年龄可能相差±0.5天。有必要使用足够数量的怀孕雌性来获得足够的胚胎进行分析。

食谱

  1. 0.1M磷酸盐缓冲盐水溶液(PBS)
    0.02 M KH 2 PO 4 ,0.04 M Na 2 HPO 4 ·2H 2 ddH 2 O中的O和0.1567 M NaCl
  2. 氨苄西林溶液
    在0.1M PBS中0.1mg / ml氨苄青霉素
  3. 0.9%氯化钠溶液
    ddH 2 O中的0.9%NaCl
  4. 1%利多卡因溶液
    1%利多卡因在0.9%NaCl溶液中
    注意:在溶液中加入少许盐酸以帮助溶解。
  5. 10%水合氯醛溶液
    10%水合氯醛在0.9%NaCl溶液中
  6. 0.2 M磷酸盐缓冲液(PB)
    0.04 M NaH 2 PO 4 和0.2 M Na 2 HPO 4 ·2H 2 d在ddH 2 O
  7. 0.05M Tris缓冲盐水(TBS)
    0.0123 M Tris,0.0377 M Tris-HCl,0.1538 M NaCl,ddH 2 O
  8. 0.5%/ 0.05%Triton溶液
    TBS中0.5%/ 0.05%Triton X-100
  9. CTB解决方案
    用10μl二甲基亚砜(DMSO)溶解500μgCTB固体粉末,然后加入230μl0.9%氯化钠溶液
  10. 4%多聚甲醛溶液
    PB中4%PFA
  11. 30%蔗糖溶液
    PB中含30%蔗糖
  12. DAPI解决方案
    ddH 2 O中1 mg / ml DAPI用于储存
    1:3,000稀释在TBS中以达到工作浓度

致谢

作者声明没有竞争利益。该方案改编自Diao 等人(2018)。我们感谢以下资助机构支持这项工作:中国NSF(31421091和31422025),青年1000计划和中华人民共和国科学技术部(2015AA020512)。

参考

  1. Diao,Y.,Cui,L.,Chen,Y.,Burbridge,T.J.,Han,W.,Wirth,B.,Sestan,N.,Crair,M。C. and Zhang,J。(2018)。 皮质和丘脑之间的相互关联有助于视网膜轴突靶向背外侧膝状体核。 Cereb Cortex 28(4):1168-1182。
登录/注册账号可免费阅读全文
  • 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: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。