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Preparation of Teased Nerve Fibers from Rat Sciatic Nerve
利用大鼠坐骨神经分离制备单根神经纤维   

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Abstract

Compared to tissue sectioning techniques, the technique of teasing single nerve fibers provides a better way to understand the structures of myelin sheaths and axons of the peripheral myelinated nerves. This protocol describes a method for preparation of teased single nerve fibers from rat sciatic nerve. In this protocol, fixed nerves are teased into single individual fibers and arranged onto adhesion microscope slides for further immuno-staining.

Keywords: Peripheral nerve(外周神经), Single nerve fiber(单神经纤维), Tease(分离), Myelin(髓磷脂), Axon(轴突), Immuno-staining(免疫染色)

Background

Schwann cells in the peripheral nervous system wrap around axons to form insulated myelin sheaths that allow the rapid conduction of action potentials. The remarkable multi-layered myelin sheath consists of elaborate structures including compact sheath, Schmidt-Lanterman incisures, Cajal bands, inner and outer mesaxons, as well as the structures in the paranodal region. To elucidate the normal and abnormal structures of myelinated fibers of peripheral nerves, teased nerve fibers are required. Methods of teasing fibers have been widely applied in studies on peripheral nerves of human and rodents. In this protocol, we describe a method of teasing peripheral nerves into single fibers for further morphological studies on the axons or myelin of peripheral nerves.

Materials and Reagents

  1. Adhesion microscope slides (CITOTEST LABWARE MANUFACTURING, catalog number: 80312-3161-16 )
    Note: These slides with special treatment process that electrostatically adheres tissue to the glass without the need for adhesives or protein coatings.
  2. Cover slips (CITOTEST LABWARE MANUFACTURING, catalog number: 80342-1130 )
  3. Cell culture dish (100 x 10 mm) (Corning, catalog number: 430167 )
  4. Animal: 4-month-old Sprague Dawley (SD) rat
  5. Chloral hydrate (Sinopharm Chemical Reagent, catalog number: 30037517 )
  6. Sodium chloride (NaCl) (Guangdong Guanghua Sci-Tech, catalog number: 1.01307.040 )
  7. Paraformaldehyde (PFA) (Guangdong Guanghua Sci-Tech, catalog number: 1.17767.014 )
  8. Phosphate-buffered saline (PBS) (Beyotime Biotechnology, catalog number: C0221A )
  9. Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
  10. Gelatin (Sigma-Aldrich, catalog number: G7041 )
  11. Anti-S100 Protein antibody, clone 15E2E2, produced in mouse (S100) (Merck, catalog number: MAB079-1 )
  12. Anti-Neurofilament 200 antibody produced in rabbit (NF) (Sigma-Aldrich, catalog number: N4142 )
  13. Alexa Fluor® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11001 )
  14. Alexa Fluor® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, InvitrogenTM, catalog number: A-11011 )
  15. TWEEN® 20 (Sigma-Aldrich, catalog number: P1379 )
  16. 4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
  17. Mounting medium for fluorescence (Vector Laboratories, catalog number: H-1000 )
  18. 10% chloral hydrate (see Recipes)
  19. 0.9% NaCl (see Recipes)
  20. 4% PFA (see Recipes)
  21. 0.1% Triton X-100 (see Recipes)
  22. Blocking buffer (see Recipes)
  23. PBST (see Recipes)
  24. 1,000x DAPI (see Recipes)

Equipment

  1. Dissecting scissors and forceps (see Figure 1A)
  2. Perfusion pump (Longer, catalog number: BT300-1F )
  3. Spring scissors (66 Vision Tech, catalog number: 54053B ) (see Figure 2A)
  4. Fine forceps (Fine Science Tools, Dumont, model: #5SF, catalog number: 11252-00 , see Figure 2A)
  5. Black wet chamber (Leibusi, catalog number: 340012 )
    Note: The wet chamber (Leibusi, catalog number: 340012 ) was made by a local workshop.
  6. Stereomicroscope (Olympus, catalog number: SZ61 )

Procedure

  1. Anaesthetize a rat with an intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g) (see Recipes).
    Note: Animal use was approved by the Southern Medical University Animal Care and Use Committee and the experimental procedure was performed in accordance with the guidelines for the ethical treatment of animals. All efforts were made to minimize animal sacrifice and suffering.
  2. With big scissors and forceps, expose the heart by cutting the thoracic cavity open.
  3. Using a perfusion pump, perfuse the rat with cold 0.9% NaCl (see Recipes) by transcardial perfusion to clear the blood in the body.
  4. Perfuse the rat with cold 4% PFA (see Recipes) to fix the rat till it becomes stiff.
  5. With big scissors and forceps, cut the skin to expose the biceps femoris and the intermuscular septum (see Figure 1B).
  6. With small scissors and forceps, separate the biceps femoris along the intermuscular septum to expose the sciatic nerve (see Figure 1C).


    Figure 1. Expose the sciatic nerve. A. Scissors and forceps used to dissect sciatic nerves; B. The biceps femoris (BF) and the intermuscular septum (IS) are shown after removal of the skin. C. Sciatic nerve is exposed after dissecting the biceps femoris. C’. A magnified image of C shows the sciatic nerve before removal of it from the rat.

  7. Use small scissors to cut the nerve at the knee and the sciatic notch, obtain the nerve and store in 4% PFA at 4 °C overnight for post-fixation.
  8. Use spring scissors to cut a nerve segment of 3-5 mm from the fixed nerve, and place the segment in a dish filled with PBS at room temperature (RT) (see Figure 2B).
  9. Use fine forceps to remove the epineurium wrapping the nerve under a stereomicroscope (see Figure 2C).
    Note: The epineurium is the outermost layer of dense irregular connective tissue surrounding the nerve. It appears more transparent than the nerve fasciculus after the fixation.
  10. Transfer the epineurium-free nerve onto an adhesion slide immersed in PBS in a dish (see Figure 2D).
  11. Use fine forceps to tease the nerve into parts under the stereomicroscope and carefully isolate the single individual fibers (see Figure 2E).
    Note: While teasing the nerve into parts, some fibers will dissociate from the nerve fasciculus individually or in small groups. Then isolate the dissociated fibers from the fasciculus and carefully tease them into single individual fibers without breaking them. Isolated fibers floating in PBS appear white in color and semi-transparent.
  12. Use fine forceps to carefully brush the single individual nerve fibers onto the adhesion microscope slide (see Figure 2F).
    Note: Good adhesion of fibers to slides is essential in this step, and make sure the fibers are well adhered to the slides. The whole fiber attaching to the slide without any detachment suggests good adherence.


    Figure 2. Step-by-step procedures of teasing individual fibers. A. Spring scissors and forceps used to tease nerve; B. A nerve segment before the removal of epineurium; C. Nerve epineurium (arrow) is half stripped off from the nerve and the epineurium-free nerve fasciculus (arrowhead). D. Nerve fasciculus immersed in PBS on an adhesion slide in a dish under a stereomicroscope; E. Nerve fasciculus with single individual fibers; F. Straight single individual nerve fibers adhering to the slide.

  13. Lift the slide out of PBS and absorb the liquid left on the slide with absorbent paper.
  14. Air-dry the adhering fibers and store at 4 °C overnight for stronger adhesion.
  15. Immuno-stain the teased fibers (steps 16-26) in the following days as soon as possible for further analysis.
    Note: Store slides at -80 °C for long-term storage. However, immunofluorescent staining performed in the next day usually gets the most guaranteed results.
  16. Wash the fibers on the slides 3 times (10 min each) with PBS.
  17. Incubate the fibers with 0.1% Triton X-100 (see Recipes) in PBS for 30 min to penetrate the membrane.
  18. Incubate the fibers with blocking buffer (see Recipes) for 1 h at RT.
  19. Incubate the fibers with primary antibodies overnight at 4 °C.
    Note: Primary antibodies of anti-S100 (1:400, diluted in blocking buffer) to show Schwann cell cytoplasm) and anti-NF (1:800, diluted in blocking buffer) to show neuron axons in the present protocol.
  20. Wash the fibers 3 times (10 min each) with PBST (see Recipes).
  21. Incubate the fibers with secondary antibodies for 2 h at RT.
    Note: Secondary antibodies of Alexa Fluor® 488 goat anti-mouse IgG (H+L) and Alexa Fluor® 568 goat anti-rabbit IgG (H+L) are used in a dilution of 1:400 in PBST.
  22. Wash the fibers 3 times (10 min each) with PBST.
  23. Incubate the fibers with DAPI (see Recipes) of 1 μg/ml in PBS for 5 min at RT.
  24. Wash the fibers with PBST for 5 min.
  25. Mounted the fibers with mounting medium and coverslip.
  26. Capture images with a fluorescent microscope.

Data analysis

Representative immunofluorescent stained images to show the structures of teased nerve fibers in Figure 3.


Figure 3. Representative immunofluorescent stained images to show the structures of teased nerve fibers. A. A single individual nerve fiber teased from the sciatic nerve of 2-week old rats was double stained with S100 (a specific marker expressed in the cytoplasm of Schwan cells) and neurofilament (NF, a specific marker for showing axon). B. A single individual nerve fiber teased from the sciatic nerve of 4-month old rats was immunostained with S100 to show the detailed structures of Cajal bands (CB) and Schmidt-Lanterman incisures (SLI) which are only exist in adult nerve fibers. PNC, perinuclear cytoplasm; NR, node of Ranvier.
Note: Both of images were stained with DAPI to show the nuclei of Schwann cells. 

Recipes

  1. 10% chloral hydrate
    Dissolve 10 g chloral hydrate in 100 ml distilled water and store at 4 °C, protect from light, and use within 1 week after preparation
  2. 0.9% NaCl
    Dissolve 9 g NaCl in 1,000 ml distilled water, store at 4 °C, and use within 1 week after preparation
  3. 4% PFA
    Dilute 40 g PFA in 1,000 ml PBS
    Stir at 65 °C until complete dissolution, store at 4 °C, and use within 2 months after preparation
  4. 0.1% Triton X-100
    Dilute 100 μl Triton X-100 into 100 ml PBS, store at RT, and use within 6 months after preparation
  5. PBST
    Dilute 500 μl TWEEN® 20 into 1,000 ml PBS to a solution containing 0.5% TWEEN® 20, store at RT, and use within 2 months after preparation
  6. Blocking buffer
    Dissolve 0.5 g gelatin (5%, w/v) in 10 ml PBS and add 300 μl Triton X-100 (0.3%) to the buffer, store at -20 °C, and use within 6 months after preparation
  7. 1,000x 4,6-diamidino-2-phenylindole (DAPI)
    Dissolve 5 mg DAPI in 5 ml distilled water to make 1,000x stock solution of 1 mg/ml, store at -20 °C, use within 2 years after preparation, and dilute to 1x with PBS before use

Acknowledgments

This protocol is adapted from the previously published paper (Wen et al., 2017). This work was supported by the National Key Basic Research Program of China (2014CB542202 and 2014CB542205), National Natural Science Foundation of China (30973095, 81371354 & 81571182); Science and Technology Project of Guangzhou (12C32121609) and Natural Science Foundation of Guangdong Province (S2013010014697) to J Guo.

References

  1. Wen, J., Qian, C., Pan, M., Wang, X., Li, Y., Lu, Y., Zhou, Z., Yan, Q., Li, L., Liu, Z., Wu, W. and Guo, J. (2017). Lentivirus-mediated RNA interference targeting RhoA slacks the migration, proliferation, and myelin formation of Schwann cells. Mol Neurobiol 54(2): 1229-1239.

简介

与组织切片技术相比,单神经纤维的技术提供了更好的方法来了解髓鞘和外周髓鞘神经的轴突的结构。 该方案描述了一种从大鼠坐骨神经制备调节的单神经纤维的方法。 在该方案中,将固定的神经梳理成单个单独的纤维并排列在粘附显微镜载玻片上用于进一步的免疫染色。
【背景】外周神经系统中的雪旺氏细胞围绕轴突包裹以形成绝缘髓鞘,其允许动作电位的快速传导。 显着的多层髓鞘由精细的结构组成,包括紧密的鞘,施密特 - 兰特曼切口,Cajal带,内外侧mesaxons,以及在paranodal区域的结构。 为了阐明外周神经有髓纤维的正常和异常结构,需要使用切割的神经纤维。 拔牙纤维的方法已被广泛应用于人类和啮齿类动物周围神经的研究。 在这个协议中,我们描述了一种将周围神经分为单纤维的方法,用于对周围神经的轴突或髓鞘进行进一步的形态学研究。

关键字:外周神经, 单神经纤维, 分离, 髓磷脂, 轴突, 免疫染色


材料和试剂




  1. 粘合显微镜载玻片(CITOTEST LABWARE MANUFACTURING,目录号:80312-3161-16)

    注意:这些幻灯片具有特殊的处理过程,将组织静电粘附到玻璃上,而不需要粘合剂或蛋白质涂层。



  2. 封面(CITOTEST LABWARE MANUFACTURING,目录号:80342-1130)



  3. 细胞培养皿(100×10mm)(Corning,目录号:430167)



  4. 动物:4个月大的Sprague Dawley(SD)大鼠



  5. 水合氯醛(国药化学试剂,目录号:30037517)



  6. 氯化钠(NaCl)(广东光华科技,目录号:1.01307.040)



  7. 多聚甲醛(PFA)(广东光华科技,目录号:1.17767.014)



  8. 磷酸盐缓冲盐水(PBS)(Beyotime Biotechnology,目录号:C0221A)



  9. Triton X-100(Sigma-Aldrich,目录号:V900502)



  10. 明胶(Sigma-Aldrich,目录号:G7041)



  11. 抗S100蛋白抗体,小鼠中产生的克隆15E2E2(S100)(Merck,目录号:MAB079-1)



  12. 抗兔神经丝200抗体(NF)(Sigma-Aldrich,目录号:N4142)



  13. Alexa Fluor  488山羊抗小鼠IgG(H + L)(Thermo Fisher Scientific,Invitrogen  TM,目录号:A-11001)



  14. Alexa Fluor  568山羊抗兔IgG(H + L)(Thermo Fisher Scientific,Invitrogen  TM,目录号:A-11011)



  15. TWEEN ® 20(Sigma-Aldrich,目录号:P1379)



  16. 4,6-二脒基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:D9542)



  17. 荧光固定介质(Vector Laboratories,目录号:H-1000)



  18. 10%水合氯醛(见配方)



  19. 0.9%NaCl(参见食谱)



  20. 4%PFA(见配方)



  21. 0.1%Triton X-100(参见食谱)



  22. 阻塞缓冲区(见配方)



  23. PBST(见配方)



  24. 1,000x DAPI(见配方)



设备




  1. 解剖剪刀和镊子(见图1A)



  2. 灌注泵(较长,目录号:BT300-1F)



  3. 春季剪刀(66 Vision Tech,目录号:54053B)(见图2A)



  4. 精镊子(Fine Science Tools,Dumont,型号:#5SF,目录号:11252-00,参见图2A)



  5. 黑色湿室(Leibusi,目录号:340012)

    注意:湿室(Leibusi,目录号:340012)由当地的车间制作。



  6. 立体显微镜(Olympus,目录号:SZ61)




程序




  1. 麻醉大鼠腹膜内注射10%的水合氯醛(0.3ml / 100g)(参见食谱)。

    注意:动物使用由南方医科大学动物护理和使用委员会批准,实验程序按照动物伦理治疗指南进行。所有努力都是为了尽量减少动物的牺牲和痛苦。



  2. 用大剪刀和镊子,通过切开胸腔打开心脏。



  3. 使用灌注泵,用冷的0.9%NaCl灌注大鼠(参见食谱),通过心脏灌注清除体内的血液。



  4. 用冷的4%PFA(见食谱)灌注大鼠,以固定大鼠,直到它变硬。



  5. 用大剪刀和镊子切割皮肤以暴露股二头肌和肌间隔(见图1B)。



  6. 用小剪刀和镊子将股二头肌沿着肌间隔分开,以露出坐骨神经(见图1C)。





    图1.暴露坐骨神经。 A.用于解剖坐骨神经的剪刀和镊子; B.除去皮肤后,显示股二头肌(BF)和肌间隔(IS)。 C.解剖股二头肌后,观察到坐骨神经。 C'。 C的放大图像显示坐骨神经,然后将其从大鼠中取出




  7. 使用小剪刀剪下膝盖和坐骨神经缺口处的神经,获得神经,并在4℃保存4%PFA过夜,进行固定。


  8. 使用弹簧剪将固定神经切断为3-5毫米的神经节,并将其放置在室温(RT)下的PBS填充盘中(见图2B)。


  9. 使用细镊子去除在立体显微镜下包裹神经的神经节(参见图2C)。

    注意:神经节是围绕神经的致密不规则结缔组织的最外层。在固定后,它看起来比神经纤维更透明


  10. 将无神经节神经转移到浸在PBS中的粘附片上(见图2D)。


  11. 使用细镊子将神经取出立体显微镜下的部分,并仔细分离单根纤维(见图2E)。

    注意:当将神经分成几部分时,一些纤维将单独或以小组从神经束分离。然后将解离的纤维与纤丝分离,并仔细挑选成单根纤维,而不会破坏它们。漂浮在PBS中的孤立纤维显示为白色,半透明


  12. 使用细镊子仔细地将单个单个神经纤维刷到粘附显微镜载玻片上(见图2F)。

    注意:在此步骤中纤维对载玻片的良好粘合是必不可少的,并且确保纤维良好地粘附在载玻片上。连接到幻灯片上的整个光纤没有任何分离,表明良好的遵守。





    图2.分开单个纤维的分步程序。 A.弹簧剪刀和钳子用于挑逗神经; B.去除神经节之前的神经节;神经上颌神经(箭头)从神经和无神经神经筋膜(箭头)半剥离。 D.在立体显微镜下将沉浸在PBS中的神经筋膜放在盘中的粘附载玻片上; E.单个纤维的神经纤维; F.直接单个单个神经纤维粘附在幻灯片上。





  13. 将玻片从PBS中取出,用吸收纸吸收载玻片上残留的液体。



  14. 空气干燥附着的纤维,并在4℃下保存过夜,以增强粘附力。



  15. 在随后的几天内尽快免疫染色细纤维(步骤16-26)以进一步分析。

    注意:将幻灯片放在-80°C进行长期存储。然而,在第二天进行的免疫荧光染色通常得到最有保证的结果。



  16. 用PBS洗涤载玻片上的纤维3次(每次10分钟)


  17. 将含有0.1%Triton X-100(参见食谱)的纤维在PBS中孵育30分钟以穿透膜。



  18. 使用封闭缓冲液(见食谱)孵育纤维1小时。



  19. 在4℃下将纤维与一抗孵育过夜。

    注意:在本方案中,抗S100(1:400,在封闭缓冲液中稀释)以显示施旺细胞细胞质的主要抗体和抗NF(1:800,在封闭缓冲液中稀释)以显示神经元轴突。 



  20. 用PBST洗涤纤维3次(每次10分钟)(参见食谱)。



  21. 在室温下将纤维与二抗孵育2小时。

    注意:在PBST中以1:400的稀释度使用Alexa Fluor 488山羊抗小鼠IgG(H + L)和AlexaFluor®568山羊抗兔IgG(H + L)的二抗。 



  22. 用PBST洗涤纤维3次(每次10分钟)。



  23. 在PBS中用DAPI(见食谱)培养1μg/ ml纤维5分钟


  24. 用PBST洗涤纤维5分钟


  25. 用安装介质和盖玻片安装光纤。



  26. 用荧光显微镜拍摄图像。



数据分析



代表性的免疫荧光染色图像显示了图3中妊娠神经纤维的结构




图3.代表性的免疫荧光染色图像显示了妊娠神经纤维的结构。 A.将来自2周龄大鼠坐骨神经的单个单独的神经纤维用S100(特异性标记物)双重染色在Schwan细胞的细胞质中表达)和神经丝(NF,用于显示轴突的特异性标记)。 B.用S100细胞免疫染色4个月大鼠坐骨神经纤维的单个神经纤维,以显示仅存在于成年神经纤维中的Cajal带(CB)和Schmidt-Lanterman切口(SLI)的详细结构。 PNC,核周细胞质; NR,Ranvier的节点。

注意:两个图像都用DAPI染色,以显示施万细胞的核。 



食谱




  1. 10%水合氯醛

    将10克水合氯醛溶解于100ml蒸馏水中,保存在4℃,防止光照,并在制备后1周内使用



  2. 0.9%NaCl

    将9克NaCl溶于1000毫升蒸馏水中,储存于4℃,准备后1周内使用


  3. 4%PFA

    在1,000 ml PBS中稀释40 g PFA
    在65°C下搅拌直到完全溶解,储存在4°C,并在准备后2个月内使用


  4. 0.1%Triton X-100

    将100μlTriton X-100稀释到100ml PBS中,在室温下储存,并在制备后6个月内使用



  5. PBST

    将500μlTWEEN ® 20稀释到1,000ml PBS中至含有0.5%TWEEN  20的溶液中,在室温下储存,并在制备后2个月内使用



  6. 阻塞缓冲区

    将0.5 g明胶(5%,w / v)溶于10ml PBS中,加入300μlTriton X-100(0.3%)至缓冲液中,储存于-20°C,准备后6个月内使用。


  7. 1,000x4,6-二脒基-2-苯基吲哚(DAPI)

    将5mg DAPI溶解在5ml蒸馏水中,制备1mg / ml的1,000x储备溶液,储存于-20℃,制备后2年内使用,并在使用PBS前用PBS稀释至1x。


致谢



该协议是从以前发表的文章(Wen等人,2017年)中改编的。 这项工作得到了国家重点基础研究计划(2014CB542202和2014CB542205),国家自然科学基金(30973095,81371354& 81571182)的支持。 广州科技项目(12C32121609)和广东省自然科学基金(S2013010014697)至郭国。



参考




  1. Wen,J.,Qian,C.,Pan,M.,Wang,X.,Li,Y.,Lu,Y.,Zhou,Z.,Yan,Q.,Li,L.,Liu,Z., Wu,W.and Guo,J.(2017)。 靶向RhoA的慢病毒介导的RNA干扰减轻了施旺细胞的迁移,增殖和髓鞘形成。 Mol Neurobiol  54(2):1229-1239。


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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wen, J., Li, L., Tan, D. and Guo, J. (2017). Preparation of Teased Nerve Fibers from Rat Sciatic Nerve. Bio-protocol 7(19): e2572. DOI: 10.21769/BioProtoc.2572.
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