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Mouse Cochlear Whole Mount Immunofluorescence
小鼠耳蜗整装包埋免疫荧光法   

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Abstract

This protocol comprises the entire process of immunofluorescence staining on mouse cochlea whole mount, starting from tissue preparation to the mounting of the tissue. This technique provides “three-dimensional” views of the stained components in order to determine the localization of a protein of interest in the tissue in its natural state and environment.

Keywords: Inner ear(内耳), Cochlea whole mount(耳蜗整装), Immunofluorescence(免疫荧光), Myosin 7a stain(肌球蛋白7a染色), Vglut3 stain(VGLUT3染色)

Materials and Reagents

  1. Ketamine hydrochloride (100 mg/ml) (Ketaset FORT DODGE)
  2. Xylazine hydrochloride (100 mg/ml) (Xylazine AnaSed)
  3. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
  4. Sodium Phosphate monohydrate (NaH2PO4.1H2O) (Sigma-Aldrich, catalog number: S9638 )
  5. Sodium Phosphate heptahydrate (NaH2PO4.7H2O) (Sigma-Aldrich, catalog number: S9390 )
  6. Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359 )
  7. Sodium Chloride (NaCl) (Sigma-Aldrich, catalog number: S3014 )
  8. Phosphate buffer (PB)
  9. Phosphate buffer saline (PBS)
  10. EDTA (Sigma-Aldrich, catalog number: E7889 )
  11. Blocking buffer
  12. Normal goat serum (NGS) (Sigma-Aldrich, catalog number: G-6767 )
  13. Saponin (Sigma-Aldrich, catalog number: 47036 )
  14. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  15. Primary Antibodies
    1. Rabbit anti-myosin VIIa antibody (a hair cell-specific marker) (Proteus Biosciences, catalog number: 25-6790 ) at a dilution of 1:50 in blocking buffer.
    2. Guinea pig anti- VGLUT3 (Vesicle Glutamate Transporter 3) antibody at 1:5,000 in blocking buffer (a gift from Dr. Robert Edward, Department of Neurology, UCSF)
  16. Secondary Antibodies
    1. Cy2-conjugated Goat anti-rabbit secondary IgG antibody (Jackson ImmunoResearch) at a dilution of 1:4,000 in PBS.
    2. Cy3-conjugated Goat anti-guinea pig secondary IgG antibody (Jackson ImmunoResearch) at a dilution of 1:4,000 in PBS.
      Note: secondary antibodies should be diluted in PBS (not blocking buffer).
  17. DAPI to counterstain nuclei (1.5 μg/ml in PBS) (Sigma-Aldrich, catalog number: D9564 ).
  18. Antifade FuorSave reagent (Calbiochem, catalog number: 34589 )
  19. 10x PB (see Recipes)
  20. 10x PBS (see Recipes)
  21. 4% PFA-PB (50 ml) (see Recipes)
  22. Blocking buffer (see Recipes)

Equipment

  1. Syringe (1 ml syringe 25G1) (BD Bioscience, catalog number: 329622 )
  2. 0.5 ml microcentrifuge tube’s caps (Thermo Fisher Scientific, catalog number: 02-681-311 )
  3. Microdissecting scissor (Fine Science Tools, catalog number: 15002-08 )
  4. Fine forceps (Ted Pella, catalog number: 5621 )
  5. Surgical blade (Feather)
  6. 5 ml Vials (Denville, catalog number: T8200-S )
  7. Pasteur pipettes (used to rinse the tissue) (Thermo Fisher Scientific, catalog number: 13-678-20 )
  8. SuperFrost slides (Thermo Fisher Scientific, catalog number: 12-550 )
  9. Cover slips (Thermo Fisher Scientific, catalog number: 12-548-5A )
  10. Petri dish (BD Biosciences, Falcon®, catalog number: 1007 )
  11. Aluminum foil
  12. Refrigerator
  13. Rocking platform
  14. Dissecting microscope
  15. Confocal microscope

Procedure

  1. Cochlea extraction and perfusion
    1. Anesthetize mice (mice overdosed with 200 μl of a mixture of ketamine hydrochloride, 100 mg/ml and xylazine hydrochloride 10 mg/ml, per 100 g of mouse weight).
    2. Pinch the toes to check if the animal is fully asleep then decapitate the mouse.
    3. Quickly and carefully remove cochleae from skull and put them in a Petri dish as follow:
      1. Cut the mouse head in half.
      2. Remove brain.
      3. Hold head on side so the 'bowl' of the skull is facing up.
      4. The temporal bone is in the back and is not continuous to the skull (there are many small bones attaching it to the skull).
      5. Push in the junctions between the skull and the temporal bone (away from the cochlea area) to break it away from skull.
      6. Carefully dissect extraneous bones away from the cochlea.
        The following video by Parker et al. (2010) shows, between min 3 and 4, how to dissect the temporal bone (cochlea) from a P3-5 mouse. The same procedure is done for an older mouse, but the only difference is the bone cochlea becomes harder.
        http://www.jove.com/video/1685/primary-culture-plasmid-electroporation-murine-organ
    4. Use a syringe to push approximately 0.5 ml of ice cold 4% PFA-PB through the round and oval windows (RW and OW) after having made a small hole in the apex of the cochlea (for orientation refer to the whole cochlea picture with legends in Figure 1 (Glen et al., 2008)).


      Figure 1. Shows a mouse decalcified left temporal bone. This labeled picture helps for orientation on the mouse anatomy of the whole temporal bone needed for good success in reproducing this protocol [this picture was borrowed from MacDonald et al. (2008) paper].

      Note: To have a very good fixation, the round window membrane should be punctured and the stapes should be removed before cochlea perfusion.
    5. Post-fix the cochleae in 5 ml 4% PFA-PB for 2 h at 4 °C under gentle agitation.
    6. Using 1 ml syringe rinse cochlea by perfusing 3 times PB through the RW and OW.
    7. Wash 3 x 15 min in fresh PB at 4 °C under gentle agitation.
  2. Cochlea decalcification and organ of Corti dissection into surface preparation
    1. Transfer the cochleae to 5 ml tube containing 5% EDTA and incubate at 4 °C under gentle agitation.

      Post-natal Age
      < P7
      P7-P15
      P15-P21
      P21
      Time in 5% EDTA
      1-2 h
      2-4 h
      5-8 h
      12- 24 h

      Note: Before going to next step, you need to check if the cochleae are very well decalcified.  Insufficient decalcification will not help to get an intact and good organ of Corti. A good decalcification is when the cochleae become soft at touch with forceps and when the cochlea bone becomes transparent.  A picture of decalcified cochlea is present in the following video.
      http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/
    2. Wash 3 x 15 min in fresh PB at 4 °C under gentle agitation.
    3. The otic capsule, the lateral wall, Reissner’s membrane and tectorial membrane should be removed in that order and then dissect the organ of Corti (for orientation on the cochlea anatomy see the cochlea mid-turn cross section Figure 2).


      Figure 2. Shows a mid-modular cross section of the mouse cochlea turn stained with toluidine blue. This labeled 20x view section helps for orientation on the mouse cochlea anatomy needed in this protocol for good success in reproducing this protocol.

    4. The remaining organ of Corti was further dissected into a surface preparation and microdissected into individual turns.
      Note: The video of Liberman's laboratory shows a method of surface preparation
      dissection that better preserves the overall structure of the organ of Corti.
      http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/
  3. Immunofluorescence
    1. Microdissected cochlea individual turns were then placed in a 0.5 ml Ependorf tube cap containing blocking buffer and incubated for 1 h at room temperature (RT) on a rocking platform.
    2. Incubate in primary antibodies diluted in blocking buffer overnight at 4 °C on a rocking platform.
      Note: Putting the cap with cochlea turns in a small Petri dish and wrapping them with parafilm will prevent dehydration.
    3. Rinse 3 x 15 min in PBS.
    4. Incubate with secondary antibodies in PBS for 2 h at RT on a rocking platform.
      Note: Because the secondary antibodies are sensitive to light, the tissue should be wrapped with aluminum foil to protect it from light.
    5. Rinse 3x 15 min in PBS.
    6. Stain the nuclei with DAPI in PBS for 15 min at RT.
    7. Rinse 3 x 15 min in PBS.
    8. Mount the cochlea whole mount turn carefully onto slides.
      Note: The orientation is very important in mounting the whole mount organ of Corti. The hair cells stereocilia should face up and be in contact with the coverslip, while the basilar membrane labeled green in Figure 2 should be on the bottom and be in contact with the slide. The video of Liberman's laboratory shows the correct orientation of the cochlea surface preparation a slide of the video is presented in Figure 3.


      Figure 3. Shows a slide of the video of Liberman's laboratory. http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/” presenting the correct orientation and preparation of the whole mount organ of Corti. The hair cells stereocilia should face up and be in contact with the coverslip, while the basilar membrane labeled green in Figure 2 should be on the bottom and be in contact with the slide. At this step if the orientation of the cochlea surface preparation is done properly it provides very good views of the stained components under the microscope.

    9. Apply a drop of antifades solution, coverslip and then let it dry.
    10. Slides are now ready to be observed under a microscope equipped with epifluorescence.
      Note: For better images use a confocal immunofluorescence microscope and do not expose your slides too much to light to avoid stain bleaching.
    11. Figure 2 in Akil et al. (2012) Neuron paper is a sample of the Mouse Cochlear Whole Mount Immunofluorescence.

Recipes

  1. 10x PB
    Dissolve 0.23 g anhydrous NaH2PO4 and 2.17 g Na2HPO4 heptahydrate into 80 ml ddH2O. Adjust pH to 7.4 and bring the final volume to 100 ml
  2. 10x PBS
    Dissolve 0.23 g anhydrous NaH2PO4 and 2.17 g Na2HPO4 heptahydrate, and 8.75 g NaCl into 80 ml ddH2O. Adjust pH to 7.4 and bring the final volume to 100 ml
  3. 4% PFA-PB (50 ml)
    Dissolve 2 g PFA in approximately 40 ml in ddH2O
    (Note: 1-You will need to heat the water and add NaOH to dissolve the PFA. 2- Keep the temperature below 60 °C during the entire process to avoid breakdown of the PFA polymers)
    After the PFA is dissolved, add 5 ml of 10x PB. Cool to RT and adjust the pH to 7.4. Bring the final volume to 100 ml. Filter and store at 4 °C
    Note: Use this solution within a week or less
  4. Blocking buffer
    20% normal goat serum (NGS) with 0.3% TritonX-100 and 0.3% Saponin in PBS

Acknowledgments

We acknowledge the support of this work by Hearing Research Inc and NIH R21 DC012118-01. We thank Neuron Journal and the Mass Eye and Ear for some illustrating images we used in this paper.

References

  1. Akil, O., Seal, R. P., Burke, K., Wang, C., Alemi, A., During, M., Edwards, R. H. and Lustig, L. R. (2012). Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron 75(2): 283-293. 
  2. MacDonald, G. H. and Rubel, E. W. (2008). Three-dimensional imaging of the intact mouse cochlea by fluorescent laser scanning confocal microscopy. Hear Res 243(1-2): 1-10.
  3. Parker, M., Brugeaud, A., Edge, A. S. B. (2010). Primary culture and plasmid electroporation of the murine organ of corti. J Vis Exp (36) e1685.

简介

该协议包括从小鼠耳蜗整体上的免疫荧光染色的整个过程,从组织制备开始到组织的安装。 该技术提供染色组分的"三维"视图,以便确定感兴趣蛋白质在其自然状态和环境中在组织中的定位。

关键字:内耳, 耳蜗整装, 免疫荧光, 肌球蛋白7a染色, VGLUT3染色

材料和试剂

  1. 盐酸氯胺酮(100mg/ml)(Ketaset FORT DODGE)
  2. 甲苯噻嗪盐酸盐(100mg/ml)(甲苯噻嗪类)
  3. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:P6148)
  4. 磷酸二氢钠一水合物(NaH 2 PO 4 PO 4·1/2H 2 O)(Sigma-Aldrich,目录号:S9638 )
  5. 磷酸钠七水合物(NaH 2 PO 4 PO 4,7H 2 O 2)(Sigma-Aldrich,目录号:S9390 )
  6. 氢氧化钠(NaOH)(Thermo Fisher Scientific,目录号:BP359)
  7. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014)
  8. 磷酸盐缓冲液(PB)
  9. 磷酸盐缓冲液(PBS)
  10. EDTA(Sigma-Aldrich,目录号:E7889)
  11. 阻塞缓冲区
  12. 正常山羊血清(NGS)(Sigma-Aldrich,目录号:G-6767)
  13. 皂苷(Sigma-Aldrich,目录号:47036)
  14. Triton X-100(Sigma-Aldrich,目录号:T8787)
  15. 原发性抗体
    1. 兔抗肌球蛋白VIIa抗体(毛细胞特异性标记)(Proteus Biosciences,目录号:25-6790)以1:50的稀释度在封闭缓冲液中。
    2. 豚鼠抗VGLUT3(囊泡谷氨酸转运蛋白3)抗体以1:5000在封闭缓冲液中(来自Dr. Robert Edward,Department of Neurology,UCSF的礼物)
  16. 二抗
    1. Cy2缀合的山羊抗兔二级IgG抗体(Jackson ImmunoResearch)以1:4,000的PBS稀释。
    2. Cy3缀合的山羊抗豚鼠第二IgG抗体(Jackson ImmunoResearch)以1:4,000的PBS稀释。
      注意:次级抗体应在PBS(不是封闭缓冲液)中稀释。
  17. DAPI复染色核(PBS中1.5μg/ml)(Sigma-Aldrich,目录号:D9564)。
  18. Antifade FuorSave试剂(Calbiochem,目录号:34589)
  19. 10x PB(参见配方)
  20. 10x PBS(见配方)
  21. 4%PFA-PB(50ml)(参见配方)
  22. 阻止缓冲区(参见配方)

设备

  1. 注射器(1ml注射器25G1)(BD Bioscience,目录号:329622)
  2. 0.5ml微量离心管帽(Thermo Fisher Scientific,目录号:02-681-311)
  3. 微切割剪(Fine Science Tools,目录号:15002-08)
  4. 细镊子(Ted Pella,目录号:5621)
  5. 手术刀(羽毛)
  6. 5ml Vials(Denville,目录号:T8200-S)
  7. 巴斯德移液管(用于冲洗组织)(Thermo Fisher Scientific,目录号:13-678-20)
  8. SuperFrost载玻片(Thermo Fisher Scientific,目录号:12-550)
  9. 盖玻片(Thermo Fisher Scientific,目录号:12-548-5A)
  10. 培养皿(BD Biosciences,Falcon ,目录号:1007)
  11. 铝箔
  12. 冰箱
  13. 摇台
  14. 解剖显微镜
  15. 共聚焦显微镜

程序

  1. 耳蜗提取和灌注
    1. 麻醉小鼠(小鼠过量用200μl氯化酮盐酸盐,100mg/ml和盐酸赛拉嗪10mg/ml的混合物/100g小鼠重量)。
    2. 捏住脚趾,检查动物是否完全睡着,然后断开鼠标。
    3. 快速小心地从颅骨中取出耳蜗,并将它们放在培养皿中如下:
      1. 将鼠标头切成两半。
      2. 删除大脑。
      3. 抓住头部,使头骨的"碗"朝上。
      4. 颞骨在后面,并且不连续到颅骨(有许多小骨头连接到头骨)。
      5. 推入颅骨和颞骨(远离耳蜗区域)之间的连接处,以将其从头骨上分离。
      6. 小心地分离远离耳蜗的外来骨骼。
        Parker等人(2010)的以下视频显示,在min 3和4之间,如何解剖来自P3-5小鼠的颞骨(耳蜗)。对于较老的小鼠进行相同的程序,但是唯一的区别是骨骼耳蜗变得更硬。
        http://www.jove.com/video/1685/primary-culture-plasmid-electroporation-murine-organ
    4. 在耳蜗的顶点做了一个小孔后,使用注射器通过圆形和椭圆形窗口(RW和OW)推动大约0.5毫升冰冷的4%PFA-PB(为了方向,参考整个耳蜗图片与传说在图1中(Glen等人,2008))

      图1.显示鼠标脱钙左颞骨。 此标记的图片有助于整个颞骨的鼠标解剖结构的取向,在重现此协议方面取得了良好的成功[此图片来自MacDonald等人(2008年)的论文]。

      注意:为了有一个非常好的固定,圆窗膜应该被刺穿,并应在耳蜗灌注之前去除镫骨。
    5. 将耳蜗在5 ml 4%PFA-PB中在4°C轻轻搅拌下固定2小时。
    6. 使用1ml注射器通过RW和OW灌注3次PB来冲洗耳蜗
    7. 在4℃下,在温和搅拌下,在新鲜PB中洗涤3×15分钟
  2. 耳蜗脱钙和Corti解剖器官成表面准备
    1. 将耳蜗转移到含有5%EDTA的5ml管中,并在4℃,温和搅拌下孵育
      产后年龄
      < P7
      P7-P15
      P15-P21
      P21
      5%EDTA时间
      1-2小时
      2-4小时
      5-8小时
      12- 24小时

      注意:在进行下一步之前,您需要检查耳蜗是否很好地脱钙。 不足的脱钙不会帮助获得完整和好的Corti器官。 良好的脱钙是当耳蜗与镊子接触时和耳蜗骨变得透明时变软。 以下视频中显示了已脱钙耳蜗的图片。
      http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/
    2. 在4℃下,在温和搅拌下,在新鲜PB中洗涤3×15分钟
    3. 耳囊,侧壁,Reissner膜和胸膜应按顺序移除,然后解剖Corti的器官(对于耳蜗解剖的取向,参见耳蜗中匝横截面图2)。


      图2.显示用甲苯胺蓝染色的小鼠耳蜗转的中间模块横截面。 这个标记的20x视图部分有助于在此协议中所需的鼠标耳蜗解剖结构的方向,以重现此协议的成功。

    4. 将Corti的剩余器官进一步解剖成表面制剂,并显微切割成单个匝 注意:Liberman实验室的视频显示了表面准备的方法

      http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/
  3. 免疫荧光
    1. 然后将显微解剖的耳蜗单匝置于含有封闭缓冲液的0.5ml Ependorf管盖中,并在摇摆平台上在室温(RT)温育1小时。
    2. 在摇动平台上在4℃下在封闭缓冲液中稀释的一抗中孵育过夜 注意:将耳蜗旋转盖放在小培养皿中,并用石蜡膜包装,可以防止脱水。
    3. 在PBS中冲洗3×15分钟。
    4. 与在PBS中的二抗在室温下在摇摆平台上孵育2小时。
      注意:由于第二抗体对光敏感,所以组织应该用铝箔包裹以防止光照。
    5. 在PBS中冲洗3x 15分钟。
    6. 在室温下用DAPI在PBS中染色细胞核15分钟
    7. 在PBS中冲洗3×15分钟。
    8. 安装耳蜗整个安装转小心幻灯片。
      注意:在安装Corti的整个装配机构时,方向是非常重要的。毛细胞毛细胞静纤毛应该面向上并且与盖玻片接触,而图2中标记为绿色的基底膜应该在底部并且与载玻片接触。利伯曼实验室的视频显示了耳蜗表面准备的正确方向,视频幻灯片如图3所示。


      图3.显示Liberman实验室视频的幻灯片。 " http://www.masseyeandear.org/research/ent/eaton-peabody/epl-histology-resources/video-tutorial-for-cochlear-dissection/"介绍整个座位的正确方向和准备科尔蒂器官。毛细胞毛细胞静纤毛应该面向上并且与盖玻片接触,而图2中标记为绿色的基底膜应该在底部并且与载玻片接触。在此步骤如果耳蜗表面的方向 准备工作正常,它提供了在显微镜下的染色组件的非常好的视图
    9. 应用一滴抗褪色溶液,盖玻片,然后让它干燥。
    10. 现在准备在装备有落射荧光的显微镜下观察载玻片。
      注意:为了获得更好的图像,请使用共聚焦免疫荧光显微镜,并且不要将幻灯片过多暴露在光线下以避免染色。
    11. 图2在Akil等人中( 2012)神经元纸是鼠标人耳蜗全 免疫荧光

食谱

  1. 10x PB
    将0.23g无水NaH 2 PO 4和2.17g Na 2 HPO 4七水合物溶于80ml ddH 2 O。调节pH至7.4,使最终体积为100 ml
  2. 10x PBS
    将0.23g无水NaH 2 PO 4和2.17g Na 2 HPO 4·7H水溶液和8.75g NaCl溶解在80ml ddH 2 O。调节pH至7.4,使最终体积为100 ml
  3. 4%PFA-PB(50ml) 将2g PFA溶解在约40ml ddH 2 O 2中 (注意:1 - 您需要加热水并加入NaOH溶解PFA。2-在整个过程中保持温度低于60℃,以避免PFA聚合物的破坏)
    在PFA溶解后,加入5ml 10×PB。冷却至室温,并将pH调节至7.4。使最终体积为100 ml。过滤并储存在4°C
    注意:在一周或更短时间内使用此解决方案
  4. 阻塞缓冲区
    含有0.3%TritonX-100和0.3%皂苷的PBS的20%正常山羊血清(NGS)

致谢

我们承认听力研究公司和NIH R21 DC012118-01的这项工作的支持。 我们感谢神经元杂志和质量眼和耳朵我们在本文中使用的一些说明图像。

参考文献

  1. Akil,O.,Seal,R.P.,Burke,K.,Wang,C.,Alemi,A.,During,M.,Edwards,R.H。和Lustig,L.R。 使用病毒介导的基因治疗恢复VGLUT3敲除小鼠的听力。 神经元 75(2):283-293。
  2. MacDonald,G.H.and Rubel,E.W。(2008)。 通过荧光激光扫描共聚焦显微镜对完整小鼠耳蜗的三维成像。 em> Hear Res 243(1-2):1-10。
  3. Parker,M.,Brugeaud,A.,Edge,A.S.B。(2010)。 corti小鼠器官的原代培养和质粒电穿孔。 J Vis Exp (36)e1685。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Akil, O. and Lustig, L. R. (2013). Mouse Cochlear Whole Mount Immunofluorescence. Bio-protocol 3(5): e332. DOI: 10.21769/BioProtoc.332.
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Natalie Friedricks
USC
We forgot to rinse our samples that had been in PFA with PBS before adding the EDTA to decalcify. Do you think this will make a big difference to our samples? And if so, do you know of anything we can do to fix the problem?
6/21/2017 5:52:42 PM Reply
Omar Akil
University of California, San Francisco

If you forgot to rinse your samples from PFA there will be no negative effect to your samples. For your information some protocols use 1% PFA with EDTA when decalcifying.

6/21/2017 6:14:26 PM


Xiaoping Liang
Johns Hopkins University
I can not see the video, would you please kindly share it to me?
Thank you
4/13/2017 8:21:08 PM Reply
Omar Akil
University of California, San Francisco

Thanks for your interest to our protocol.
The video was on the Massachusetts Eye and Ear web site but it looks like they removed it.

4/14/2017 4:08:49 PM