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Fluorescence Microscopy for Cilia in Cultured Cells and Zebrafish Embryos
荧光显微镜法检测培养细胞和斑马鱼胚胎中的纤毛   

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Abstract

Cilia are microtubule-based hair-like projections found in organisms, ranging from protozoa to mammals. This protocol provides methods for immunofluorescence staining of cilia in cultured cells and zebrafish embryos.

Keywords: Fluorecence microcopy(荧光显微镜), Cilia(纤毛), Zebrafish(斑马鱼), Cultured cells(培养的细胞)

Materials and Reagents

  1. hTERT-RPE1 cell line (ATCC , catalog number: CRL-4000 )
  2. Zebrafish (AB strain) (China Zebrafish Resource Center)
  3. DMEM/F12 medium (Life Technologies, InvitrogenTM, catalog number: 11330-032 )
  4. Fetal bovine serum (Life Technologies, InvitrogenTM, catalog number: 16000-044 )
  5. Hygromycin B (Life Technologies, InvitrogenTM, catalog number: 10687010 )
  6. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  7. Pure ethanol (Sinopharm Chemical Regent, catalog number: 10009218 )
  8. Low-melting-point agarose (Sigma-Aldrich, catalog number: A9414 )
  9. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3912 )
  10. Triton X-100 (AMRESCO, catalog number: 0694 )
  11. Tween-20 (AMRESCO, catalog number: 0777 )
  12. Mouse monoclonal anti-acetylated tubulin antibody (Sigma-Aldrich, catalog number: T-6793 )
  13. Goat anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-546 (Life Technologies, catalog number: A11030 )
  14. Donkey anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-488 (Life Technologies, catalog number: A21202 )
  15. 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D8417 )
  16. Dako mounting medium (Dako, catalog number: s3023 )
  17. PBS (see Recipes)
  18. PBT (see Recipes)
  19. 4% paraformaldehyde (PFA) fixation solution (see Recipes)
  20. Embryo fixation buffer (see Recipes)
  21. Embryo blocking buffer (see Recipes)
  22. 4', 6-diamidino-2-phenylindole (DAPI) stock solution (see Recipes)
  23. Complete growth medium (see Recipes)
  24. Serum free medium (see Recipes)
  25. 75% ethanol (see Recipes)

Equipment

  1. Millex-GP Filter unit with 0.22 µm pore size (Millipore, catalog number: SLGP033RS )
  2. Forceps (The duMONT Company, catalog number: 0203-5/15-PO )
  3. 10 cm Petri dishes (BD Biosciences, Falcon®, catalog number: 353003 )
  4. 12-well plates (Corning, catalog number: 3513 )
  5. 2 ml Eppendorf (EP) tubes (Axygen, catalog number: MCT-200-C )
  6. Glass slides (Fan Yi, catalog number: 7105P )
  7. 18mm diameter circle cover slips (Thermo Fisher Scientific, catalog number: 12-545-84 )
  8. Parafilm (Parafilm M®, catalog number: PM-996 )
  9. 100 ml beaker (Thermo Fisher Scientific, catalog number: 1201-0100 )
  10. Filter paper (GE, catalog number: 10311611 )
  11. Plastic transfer pipette (Thermo Fisher Scientific, catalog number: 11387873 )
  12. Shakers (Qilinbeier, catalog number: TS-8S )
  13. Leica TCS SP5 confocal microscope equipped with a 63x oil objective and a 63x NA1.2 water immersion objective

Procedure

  1. Immunofluorescence labeling of cilia in cultured cells
    1. hTERT-RPE1 cells are cultured as described in the ATCC website (http://www.atcc.org/Products/All/CRL-4000.aspx#culturemethod). Briefly, cells are grown in 10 cm Petri dishes with 10-12 ml complete growth medium (see Recipes) and media are changed every other day. Cells are subcultivated at 1: 5 ratio twice a week when cell concentration reaches between 2 x 104 and 4 x 104 cells/cm2. An inoculum of 4 x 103 to 6 x 103 viable cells/cm2 is recommended.
    2. Sterilize 18 mm diameter circle cover slips by immersing in 75% ethanol in a 100 ml beaker over night. Pick cover slips by using forceps and put slips into 12-well-plate, one slip per well. Rinse slips three times with 1 ml PBS per well each time. Add 1 ml complete growth medium to each well, and then add appropriate aliquots of the dissociated cell suspension.
    3. Culture for 1-2 days until cells reach 60-90% confluence. Wash three times with 1 ml PBS per well each time to get ride of serum, then add 1 ml serum free medium and culture for 48 h to induce cilia formation. The culture time and confluence should be determined by researchers themselves to get the best result for different experiments. Generally, a confluence higher than 60% is recommended for efficient cilia induction.
    4. Before fixation, place cells on ice for 30 min to destabilize microtubules and rinse once with 1 ml ice-cold PBS for each well.
    5. Fix cells with 1 ml of freshly prepared 4% PFA in PBS per well for 10 min at room temperature (RT).
    6. Briefly rinse cells three times with 1 ml PBS each time per well, and permeabilize cells with 1 ml 0.5% TritonX-100 in PBS per well for 15 min at RT.
      Note: PFA waste should be collected in an appropriate container for proper disposal.
    7. Briefly rinse cells three times with 1 ml PBS each time per well.
    8. Incubate cells with 1 ml 1% BSA in PBS each well for 1 h at RT.
    9. Make a humid chamber with a new 10 cm Petri dish by putting a piece of wet filter paper and a piece of parafilm on the top of the paper. Make sure the parafilm is flat. Transfer the cover slips with blocked cells by using forceps onto the parafilm. Add 100 µl diluted mouse anti-acetylated tubulin antibody (1: 1,000 in PBS with 1% BSA) onto each slip and incubate for 2 h at RT or overnight at 4 °C.
    10. Wash cells with 200 µl 1% BSA in PBS per slip three times for 5 min each.
    11. Add 100 µl diluted secondary antibody, Goat anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-546 (1: 1,000 in PBS with 1% BSA) for 1 h at RT in the dark.
    12. Wash cells with 200 µl 1% BSA in PBS per slip three times for 5 min each.
    13. To stain nuclei, dilute DAPI stock solution with PBS to 0.5 µg/ml, add 100 µl diluted DAPI onto each slip and incubate for 1 min at RT.
    14. Wash cells with 200 µl PBS per slip three times for 5 min each.
    15. Mount slides with 10-20 µl Dako mounting medium as shown in Figure 1 and dry them for at least 2 h at RT in the dark. Store slides at 4 °C. Samples are good for at least 1 month, but DAPI signals will diffuse and become dim after 1 week. It’s better to take images within 1 week.
    16. Take images on a Leica TCS SP5 confocal microscope with a 63x oil objective. Optical sections are captured at 0.5 µm intervals and z-stack images are obtained by maximum intensity projections. A typical image can be found in Figure 1j of Cao et al. (2012).
     
  2. Whole mount immunofluorescence of zebrafish embryos
    1. Zebrafish (AB strain) were raised and maintained under standard conditions and staged as previously described in hours post fertilization (hpf) (please refer to The Zebrafish Book: http://zfin.org/zf_info/zfbook/zfbk.html).
    2. Dechorionate zebrafish embryos at the 8-somite stage or at the 24 hpf stage through pronase treatment or manually with forceps. For detailed method about dechorionation, please refer to Chapter 4 of The Zebrafish Book (http://zfin.org/zf_info/zfbook/chapt4/4.1.html).
    3. Transfer embryos with plastic transfer pipette into 2 ml EP tubes, up to 100 embryos each tube. Remove extra water and fix embryos with 1 ml embryo fixation buffer for 2 h at RT or overnight at 4 °C with gentle rocking at 15-20 times per minute on a shaker. All washings and incubations should be done with gentle rocking. All solutions are added and removed by using pipette with 1 ml tips. PFA and methanol waste should be collected in an appropriate container for proper disposal.
    4. After a brief rinse with 1 ml PBT, dehydrate embryos through a graded methanol/PBT series (25%, 50%, 75%, 100%). Rock the embryos gently at RT for 5 min at each step.
    5. Incubate embryos overnight in 1ml 100% methanol at -20 °C. Embryos can be stored at -20 °C for up to 1 month before further staining.
    6. Rehydrate embryos in a methanol/PBT graded series (75%, 50%, 25%, 5 min each at RT). Rinse three times with PBT for 5 min each at RT, and use 1 ml PBT each time per tube.
    7. Incubate embryos for 1-2 h in 1 ml embryo blocking buffer at RT.
    8. Incubate samples overnight at 4 °C with mouse anti-acetylated tubulin antibody diluted in 300-500 µl blocking buffer (1: 1,000).  
      Note: At least 300 µl is recommended for sufficient incubation.
    9. Wash three times for 10 min each, with 1ml 0.5% Triton X-100 in PBS each time.
    10. Incubate embryos with 300-500 µl secondary antibody, Donkey anti-mouse IgG (H+L) antibody conjugated with Alexa Fluor-488 diluted in blocking buffer (1: 1,000) overnight at 4 °C or for 2 h at RT.
    11. Rinse three times for 10 min each with 1ml 0.5% Triton X-100 in PBS each time.
    12. To stain nuclei, dilute DAPI stock solution with PBT to 0.5 µg/ml, and incubate embryos with 1 ml diluted DAPI each tube for 5-10 min at RT. Then wash embryos three times for 5 min each with 1 ml PBT. Embryos are now ready for embedding. Otherwise, please keep stained embryos in PBT at 4 °C for up to 1 week.
    13. Embed embryos in 1% low melting agarose. Briefly, prepare a 1% low-melting-point agarose solution in H2O by heating in a microwave oven. Keep the solution liquid in a tube in a 37 °C water bath. This solution remains liquid at 37 °C, but quickly hardens when cooled lower than 30 °C. Use 35 mm culture dishes for embryos mounting and imaging. First, add 1 ml agarose solution in each dish and let it gel at RT. Make sure the agarose is flat and covers the whole bottom of the dish. Transfer four embryos by using plastic transfer pipette onto the agarose gel of each dish. Quickly pipette a small drop of agarose onto the embryo, and position the embryo at the appropriate angle in the agarose using forceps and allow the agarose to harden. Make sure the embryos are distant from each other, so that you can mount one by one. 24 hpf embryos should be mounted laterally to show the pronephric duct, and 8-somite-stage embryos should be mounted to show the Kupffer’s Vesicle on the up side. Please refer to Figure 7a and b of the Reference 1 for correct orientations. Mounted embryos should be examined at the same day.
    14. Add 2-3 ml H2O into the 35 mm dishes with mounted embryos, and take images on a Leica TCS SP5 MP confocal microscope with a 63x NA1.2 water immersion objective. Optical sections are captured at 0.5-µm intervals and z-stack images are obtained by maximum intensity projections. A typical image can be found in Figure 7g and j of Reference 1.


      Figure 1. Mounting cells for microscopy

Recipes

  1. PBS
    137 mM NaCl
    2.7 mM KCl
    8 mM Na2HPO4
    2 mM KH2PO4
    pH 7.4
    Autoclaved for long time storage
  2. PBT
    PBS with 0.1% Tween-20
  3. 4% PFA
    Dissolve 4 g PFA in 100 ml PBS, heat to 60 °C for 1 h with stirring
    The cooled solution can be filtered through Millex-GP Filter unit with 0.22 µm pore size, and stored at -20 °C. It is good for a month at -20 °C, or use immediately after preparation, which is recommended.
  4. Embryo fixation buffer
    4% PFA in PBS supplemented with 0.5% Triton X-100
  5. Embryo blocking buffer
    2% BSA
    0.5% normal goat serum
    1% DMSO
    0.5% Triton X-100 in PBS
  6. 4', 6-diamidino-2-phenylindole (DAPI) stock solution
    Dissolve DAPI powder in dimethylformamide (DMF) to make a 5 mg/ml stock solution
  7. Complete growth medium
    DMEM/F12 medium supplemented with 10% fetal bovine serum and 0.01 mg/ml hygromycin B
  8. Serum free medium
    DMEM/F12 medium supplemented with 0.01 mg/ml hygromycin B
  9. 75% ethanol
    75 ml pure ethanol mixed with 25 ml H2O

Acknowledgments

This protocol was adapted from previous work (Cao et al., 2012; Chapter 4 of The Zebrafish Book (http://zfin.org/zf_info/zfbook/chapt4/4.1.html)). The work was supported by the National Basic Research Program of China (2012CB945003 and 2010CB912102), National Science Foundation of China (30971430, 30830060, and 31010103910), and Chinese Academy of Sciences (XDA01010107).

References

  1. Cao, J., Shen, Y., Zhu, L., Xu, Y., Zhou, Y., Wu, Z., Li, Y., Yan, X. and Zhu, X. (2012). miR-129-3p controls cilia assembly by regulating CP110 and actin dynamics. Nat Cell Biol 14(7): 697-706.

简介

纤毛是在生物体中发现的基于微管的头发样突起,从原生动物到哺乳动物。 该协议提供了在培养的细胞和斑马鱼胚胎中的纤毛的免疫荧光染色的方法。

关键字:荧光显微镜, 纤毛, 斑马鱼, 培养的细胞

材料和试剂

  1. hTERT-RPE1细胞系(ATCC,目录号:CRL-4000)
  2. 斑马鱼(AB株)(中国斑马鱼资源中心)
  3. DMEM/F12培养基(Life Technologies,Invitrogen TM ,目录号:11330-032)
  4. 胎牛血清(Life Technologies,Invitrogen TM ,目录号:16000-044)
  5. 潮霉素B(Life Technologies,Invitrogen TM ,目录号:10687010)
  6. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  7. 纯乙醇(Sinopharm Chemical Regent,目录号:10009218)
  8. 低熔点琼脂糖(Sigma-Aldrich,目录号:A9414)
  9. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3912)
  10. Triton X-100(AMRESCO,目录号:0694)
  11. Tween-20(AMRESCO,目录号:0777)
  12. 小鼠单克隆抗乙酰化微管蛋白抗体(Sigma-Aldrich,目录号:T-6793)
  13. 与Alexa Fluor-546(Life Technologies,目录号:A11030)偶联的山羊抗小鼠IgG(H + L)抗体
  14. 与Alexa Fluor-488(Life Technologies,目录号:A21202)缀合的驴抗小鼠IgG(H + L)抗体
  15. (DAPI)(Sigma-Aldrich,目录号:D8417)
  16. Dako封固剂(Dako,目录号:s3023)
  17. PBS(请参阅配方)
  18. PBT(参见配方)
  19. 4%多聚甲醛(PFA)固定溶液(参见配方)
  20. 胚胎固定缓冲液(见配方)
  21. 胚胎阻断缓冲液(参见配方)
  22. 4',6-二脒基-2-苯基吲哚(DAPI)储备溶液(参见配方)
  23. 完全生长培养基(参见食谱)
  24. 无血清培养基(见配方)
  25. 75%乙醇(见配方)

设备

  1. 具有0.22μm孔径的Millex-GP过滤器单元(Millipore,目录号:SLGP033RS)
  2. 镊子(duMONT公司,目录号:0203-5/15-PO)
  3. 10cm培养皿(BD Biosciences,Falcon ,目录号:353003)
  4. 12孔板(Corning,目录号:3513)
  5. 2ml Eppendorf(EP)管(Axygen,目录号:MCT-200-C)
  6. 玻璃载玻片(Fan Yi,目录号:7105P)
  7. 18mm直径的圆形盖玻片(Thermo Fisher Scientific,目录号:12-545-84)
  8. 石蜡膜(Parafilm M ,目录号:PM-996)
  9. 100毫升烧杯(Thermo Fisher Scientific,目录号:1201-0100)
  10. 滤纸(GE,目录号:10311611)
  11. 塑料转移移液管(Thermo Fisher Scientific,目录号:11387873)
  12. (Qilinbeier,目录号:TS-8S)
  13. Leica TCS SP5共焦显微镜,配备有63x油镜和63x NA1.2水浸物镜

程序

  1. 培养细胞中纤毛的免疫荧光标记
    1. 如ATCC网站所述培养hTERT-RPE1细胞 ( http://www.atcc.org/Products/All/CRL- 4000.aspx#culturemethod )。简而言之,  细胞在具有10-12ml完全生长的10cm培养皿中生长 介质(见Recipes)和介质每隔一天更换一次。细胞是 当细胞浓度达到时,以1:5比率每周两次传代培养  在2×10 4个细胞/cm 2和4×10 4个细胞/cm 2之间。接种物4×10 3至6× 10 3 活细胞/cm <2> 。
    2. 灭菌18 mm直径 通过浸泡在100ml烧杯中的75%乙醇中的圆形盖玻片 晚。使用镊子和盖玻片挑选盖玻片 12孔板,每孔一个。用1ml PBS冲洗卡片三次 每孔。向每个孔中加入1ml完全生长培养基,和 然后加入适当等分的解离的细胞悬浮液。
    3. 培养1-2天,直到细胞达到60-90%汇合。洗三 次,每次1ml PBS,每次取血清,然后加1 ml无血清培养基并培养48小时以诱导纤毛形成。的 培养时间和融合应由研究人员决定 自己获得不同实验的最佳结果。 一般来说,a   建议有效纤毛的汇合率高于60% 感应
    4. 固定前,将细胞置于冰上30分钟 使微管不稳定,并用1ml冰冷的PBS冲洗一次 好。
    5. 用1ml新鲜制备的PBS中的4%PFA /孔将细胞固定在室温(RT)下10分钟。
    6. 用每孔每次用1ml PBS短暂冲洗细胞三次,和 用PBS中的1ml 0.5%TritonX-100 /孔透化细胞15分钟   。
      注意:PFA废物应收集在适当的容器中以妥善处理。
    7. 用每孔每次用1ml PBS短暂冲洗细胞三次。
    8. 将细胞与PBS中的1ml 1%BSA在室温下孵育1小时
    9. 用一个新的10厘米培养皿制作一个潮湿的房间   湿滤纸和在纸的顶部的一片石蜡膜。 使 确保石蜡膜是平的。 转移带有阻塞细胞的盖玻片 通过使用镊子在石蜡膜上。 加入100μl稀释的小鼠 抗乙酰化微管蛋白抗体(在含1%BSA的PBS中1:1000)   并在室温下孵育2小时或在4℃下过夜
    10. 用200μl1%BSA的PBS溶液洗涤细胞,每次3次,每次5分钟
    11. 加入100μl稀释的二抗,山羊抗小鼠IgG(H + L) 与Alexa Fluor-546偶联的抗体(在含1%BSA的PBS中1:1000) 在室温下在黑暗中1小时。
    12. 用200μl1%BSA的PBS溶液洗涤细胞,每次3次,每次5分钟
    13. 为染色细胞核,用PBS稀释DAPI储备溶液至0.5μg/ml,加入   将100μl稀释的DAPI加到每个片上,在室温下孵育1分钟
    14. 用200μlPBS每次洗涤细胞三次,每次5分钟
    15. 用10-20μlDako固定介质装载载玻片,如图1所示 并在室温下在黑暗中干燥至少2小时。 将载玻片保存在4°C。 样品至少有1个月有效,但DAPI信号会扩散   1周后变暗。 最好在1周内拍摄图片。
    16. 用Leica TCS SP5共焦显微镜用63x油拍摄图像 目的。 以0.5μm的间隔和z堆叠捕获光学部分   通过最大强度投影获得图像。 典型的图像 可以在Cao等人的图1j(2012)中找到。
     
  2. 斑马鱼胚胎的整体免疫荧光
    1. 斑马鱼(AB菌株)在标准条件下培养和维持 条件和分阶段如前所述小时后 受精(hpf)(请参阅斑马鱼书: http://zfin.org/zf_info/zfbook/zfbk.html )。
    2. Dchorionate 斑马鱼胚胎在8体细胞阶段或24 hpf阶段通过 链霉蛋白酶处理或用镊子手动。详细方法 dechorionation,请参阅斑马鱼书的第4章( http: //zfin.org/zf_info/zfbook/chapt4/4.1.html )。
    3. 转让 胚胎用塑料移液管移入2ml EP管,达到100 胚胎每个管。删除额外的水和修复胚胎与1毫升胚胎 固定缓冲液在室温下2小时或在4°C过夜,轻轻摇动 在振荡器上以每分钟15-20次。所有洗涤和孵化 应该用轻柔的摇摆来做。添加和去除所有溶液 通过使用移液器与1毫升提示。 PFA和甲醇废物应该 收集在适当的容器中以妥善处置
    4. 用1ml PBT短暂冲洗后,通过分级脱水胚胎 甲醇/PBT系列(25%,50%,75%,100%)。在RT轻轻摇动胚胎  每步5分钟。
    5. 孵育胚胎在1ml 100%   甲醇在-20℃。 胚胎可以在-20°C储存长达1个月 然后进一步染色
    6. 在甲醇/PBT中使胚胎再水化 (75%,50%,25%,每次5分钟,RT)。 冲洗三次 PBT,每次5分钟,每管使用1ml PBT
    7. 在室温下在1ml胚胎阻断缓冲液中孵育胚胎1-2小时
    8. 孵育样品在4℃过夜与小鼠抗乙酰化微管蛋白 抗体稀释在300-500μl封闭缓冲液(1:1000)中。  
      注意:建议充分孵育至少300μl。
    9. 每次洗涤三次,每次10分钟,每次用1ml 0.5%Triton X-100的PBS
    10. 孵育胚胎与300-500微升第二抗体,驴抗小鼠   IgG(H + L)抗体与Alexa Fluor-488偶联,在封闭中稀释 缓冲液(1:1000)在4℃过夜或在室温下2小时
    11. 用PBS每次1ml 0.5%Triton X-100冲洗三次,每次10分钟
    12. 为染色细胞核,用PBT稀释DAPI储备溶液至0.5μg/ml,   孵育胚胎与1毫升稀释DAPI每管5-10分钟,在室温。 然后用1ml PBT洗涤胚胎3次,每次5分钟。 胚胎是 现在准备嵌入。 否则,请保持染色的胚胎在PBT 在4°C长达1周
    13. 嵌入胚胎在1%低熔点 琼脂糖。简言之,制备H 2 O中的1%低熔点琼脂糖溶液  通过在微波炉中加热。将溶液保持在管中的液体中  37℃水浴。该溶液在37℃下保持液体,但很快 当冷却低于30℃时硬化。使用35毫米培养皿 胚胎安装和成像。首先,在每个中加入1ml琼脂糖溶液 并让其在RT凝胶。确保琼脂糖平坦,盖住 整个底部的菜。通过使用塑料转移四个胚胎 转移到每个培养皿的琼脂糖凝胶上。快速吸移a 小滴琼脂糖到胚胎上,并将胚胎定位在 适当的角度在琼脂糖使用镊子和允许琼脂糖 硬化。确保胚胎彼此远离,让你 可以一个接一个地安装。 24 hpf胚胎应该横向安装显示  前肾管和8体细胞期胚胎应安装到 显示Kupffer的囊泡在上面。请参考图7a和图7a  b,以获得正确的方向。安装胚胎应该 在同一天进行检查。
    14. 将2-3ml H 2 O加入到35mm孔中 菜肴与安装的胚胎,并在Leica TCS SP5 MP上拍摄图像 共聚焦显微镜与63x NA1.2水浸物镜。 光学 截面以0.5-μm间隔捕获,z-stack图像 通过最大强度投影获得。 可以找到典型的图像 在参考文献1的图7g和j中

      图1.安装显微镜细胞

食谱

  1. PBS
    137 mM NaCl 2.7mM KCl
    8mM Na 2 HPO 4
    2mM KH 2 PO 4 sub/
    pH 7.4
    高压灭菌长时间存储
  2. PBT
    PBS,含有0.1%Tween-20
  3. 4%PFA
    将4g PFA溶于100ml PBS中,在搅拌下加热至60℃1小时 冷却的溶液可以通过具有0.22μm孔径的Millex-GP过滤器单元过滤,并储存在-20℃。 在-20°C下保存一个月,或者在准备后立即使用,建议使用。
  4. 胚胎固定缓冲区
    补充有0.5%Triton X-100的PBS中的4%PFA
  5. 胚胎阻断缓冲液
    2%BSA
    0.5%正常山羊血清
    1%DMSO 0.5%Triton X-100的PBS溶液中
  6. 4',6-二脒基-2-苯基吲哚(DAPI)储液
    将DAPI粉末溶解在二甲基甲酰胺(DMF)中以制备5mg/ml储备溶液
  7. 完全生长培养基
    补充有10%胎牛血清和0.01mg/ml潮霉素B的DMEM/F12培养基
  8. 无血清培养基
    补充有0.01mg/ml潮霉素B的DMEM/F12培养基
  9. 75%乙醇 75ml纯乙醇与25ml H 2 O混合

致谢

该协议改编自以前的工作(Cao等人,2012; The Zebrafish Book的第4章(http://zfin.org/zf_info/zfbook/chapt4/4.1.html))。 这项工作得到了中国国家基础研究计划(2012CB945003和2010CB912102),中国国家科学基金(30971430,30830060和31010103910)和中国科学院(XDA01010107)的支持。

参考文献

  1. Cao,J.,Shen,Y.,Zhu,L.,Xu,Y.,Zhou,Y.,Wu,Z.,Li,Y.,Yan,X.and Zhu, miR-129-3p通过调节CP110和肌动蛋白动力学来控制纤毛聚集。 Nat Cell Biol 14(7):697-706。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cao, J., Zhu, X. and Yan, X. (2014). Fluorescence Microscopy for Cilia in Cultured Cells and Zebrafish Embryos. Bio-protocol 4(14): e1188. DOI: 10.21769/BioProtoc.1188.
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