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Transmission Electron Microscopy (TEM) Protocol: Observation Details within Cells
透射电子显微镜(TEM)法观察细胞内的细胞器结构   

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Abstract

Transmission electron microscopy is a technique for observing the fine details of organelles in cells or tissues. This protocol is to be used to exam the membrane structure in cells with or without virus infection. Modifications should be made if users want to get images from tissues.

Keywords: Transmission Electron Microscopy(透射电子显微镜), TEM(TEM), Cell(细胞), Organelle(细胞器)

Materials and Reagents

  1. Trypsin: working solution is 0.5% trypsin-0.2% EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
  2. NaH2PO4 (Kanto, catalog number: 37239-01 )
  3. Na2HPO4 (Chemical, catalog number: 37240-01)
  4. Alcohol
  5. Poly/Bed 812 (Electron microscopy sciences, catalog number: 14900 )
  6. Araldite 502 (Electron microscopy sciences, catalog number: 10900 )
  7. Dodecenylsuccinic anhydride (DDSA) (TAAB, catalog number: D025 )
  8. DMP-30 (Electron microscopy sciences, catalog number: 13600 )
  9. Propylene oxide (Merk, catalog number: S4912527-745 )
    Note: Toxic, need to handle with care and collect after use.
  10. Syringe
  11. Dropper
  12. Grid (200 mesh) (TAAB)
  13. 0.1 M Phosphate buffer (PB) (see Recipes)
  14. Paraformaldehyde (4% in PB) (Bionovas, catalog number: AP0130-0500 ) (see Recipes)
  15. Osimium tetraoxide (1% in PB) (Electron microscopy sciences, catalog number: 51007 ) (see Recipes)
    Note: Toxic, need to handle with care and collect after use.
  16. Epon (see Recipes)
  17. Toluidune Blue (Sigma-Aldrich, catalog number: NO202-146-2) (see Recipes)
  18. Uranyl acetate (Art, catalog number: 8473) (see Recipes)
  19. Lead citrate (TAAB, catalog number: L003 ) (see Recipes)

Equipment

  1. Eppendorf tubes
  2. Centrifuge
  3. Vacuum drying oven
  4. Diamond knife (Diatome)
  5. Ultracut (Leica)
  6. TEM (HITACHI, model: H-7100 )

Procedure

Note: Put all droppers and syringes into the 60 °C oven the day before this procedure to remove all the water inside all stuffs.

  1. After treatment, collecting cells (1 x 106) with 0.5% trypsin-EDTA and washing cell with 0.5 ml 0.1 M phosphate buffer (PB) in an eppendorf tube by centrifuge (300 x g, 5 min).
  2. Add 0.5 ml paraformaldehyde (4% in PB) to fix cells in room temperature (RT) for at least 30 min (or store cells at 4 °C for long term storage).
  3. Wash cells 3 times with 0.5 ml 0.1 M PB by centrifuge (300 x g, 5 min).
  4. Add 0.5 ml Osimium tetraoxide (1% in PB) into the tube and incubate cells for 1 h at RT.
  5. Wash cells 3 times with 0.5 ml 0.1 M PB by centrifuge (300 x g, 5 min).
  6. Dehydrate cells with adding 0.5 ml 70% alcohol for 5 min twice→ centrifuge (300 x g, 5 min)→ remove alcohol.
  7. Dehydrate cells with adding 0.5 ml 85% alcohol for 5 min twice→ centrifuge (300 x g, 5 min)→ remove alcohol.
  8. Dehydrate cells with adding 0.5 ml 95% alcohol for 5 min for three times→centrifuge (300 x g, 5 min)→ remove alcohol.
  9. Dehydrate cells with adding 0.5 ml 100% alcohol for 10 min for five times→ centrifuge (300 x g, 5 min)→ remove alcohol.
    Note: From step 10, all materials used should be from 60 °C oven.
  10. Add 0.5 ml propylene oxide into tubes and incubate for 3 min twice at RT.→ centrifuge (300 x g, 5 min)→remove propylene oxide.
  11. Add 1 ml (propylene oxide:Epon = 1:1) into tubes and incubate for 1 h at RT.→ centrifuge (300 x g, 5 min)→ remove Propylene oxide: Epon.
  12. Add 1 ml pure Epon into tubes and put the samples into the vacuum drying oven to vacuum for 1 h at RT.→ remove Epon.
  13. Add 1 ml pure Epon into tubes and vacuum for 1 h at RT.→ remove Epon.
  14. Add 1 ml pure Epon into tubes and vacuum overnight at RT.
  15. Put tubes into 60 °C oven for at least 48 h.
  16. Trimming-remove the edge of the block.
  17. Get thick section (1 μm) with Ultracut using diamond knife→put the thick section on a slide and stain with 0.5% toluidune blue for around 40 s to 1 min (until the edge is dried)→ wash 1 min with water → observe under microscopy to find the cells.
  18. Once we found the cells on the thick section, change the thickness of Ultracut to get thin sections (65-70 nm).
  19. Put the thin sections on the grids (3-4 sections on one grid).
  20. Stain sections with uranyl acetate and lead citrate for 3 min sequentially.
  21. Wash the grids with water for 1 min and let them dry completely.
  22. Get images from digital camera on TEM with identical magnificence.

Recipes

  1. 0.1 M Phosphate buffer (PB)
    19 ml 0.2 M NaH2PO4
    81 ml 0.2 M Na2HPO4
  2. 4% paraformaldehyde
    4% paraformaldehyde in PB
  3. Osimium tetraoxide
    1% osimium tetraoxide in PB
  4. Epon
    Use syringe to mix all reagents below to make Epon.
    Poly/Bed 812 10 ml
    Araldite 502 6 ml
    DDSA 18 ml
    DMP-30 0.7 ml
    Epon could be stored in 4 °C or -20 °C for 1-2 weeks.
  5. Toluidine Blue
    0.5% Toluidine Blue
    1% Sodium borate/in H2O
    Filter with 0.22 μM filter
  6. Uranyl acetate
    Saturated uranyl acetate in 50% Alcohol
  7. Lead citrate
    1% lead citrate in H2O
    Add 3 drops of 10 M NaOH (around 0.5 ml)

Acknowledgments

This protocol is adapted from Chau and Lu (1996) and Lee et al. (2012).

References

  1. Chau, Y. P. and Lu, K. S. (1996). Differential permeability of blood microvasculatures in various sympathetic ganglia of rodents. Anat Embryol (Berl) 194(3): 259-269.
  2. Lee, C. P., Liu, P. T., Kung, H. N., Su, M. T., Chua, H. H., Chang, Y. H., Chang, C. W., Tsai, C. H., Liu, F. T. and Chen, M. R. (2012). The ESCRT machinery is recruited by the viral BFRF1 protein to the nucleus-associated membrane for the maturation of Epstein-Barr Virus. PLoS Pathog 8(9): e1002904.

简介

透射电子显微镜是用于观察细胞或组织中细胞器的细节的技术。 该协议用于检查有或没有病毒感染的细胞中的膜结构。 如果用户想要从组织获得图像,应该进行修改。

关键字:透射电子显微镜, TEM, 细胞, 细胞器

材料和试剂

  1. 胰蛋白酶:工作溶液是0.5%胰蛋白酶-0.2%EDTA(Life Technologies,Gibco ,目录号:15400-054)
  2. NaH 2 PO 4(Kanto,目录号:37239-01)

  3. (化学品,目录号:37240-01)。
  4. 酒精
  5. Poly/Bed 812(电子显微镜科学,目录号:14900)
  6. Araldite 502(电子显微镜科学,目录号:10900)
  7. 十二碳烯基琥珀酸酐(DDSA)(TAAB,目录号:D025)
  8. DMP-30(电子显微镜科学,目录号:13600)
  9. 环氧丙烷(Merk,目录号:S4912527-745)
    注意:有毒,需要小心处理并在使用后收集。
  10. 注射器
  11. 滴管
  12. 网格(200目)(TAAB)
  13. 0.1 M磷酸盐缓冲液(PB)(参见配方)
  14. 多聚甲醛(PB中4%)(Bionovas,目录号:AP0130-0500)(参见配方)
  15. 四氧化锇(1%在PB中)(电子显微镜科学,目录号:51007)(参见配方)
    注意:有毒,需要小心处理并在使用后收集。
  16. Epon(见配方)
  17. Toluidune Blue(Sigma-Aldrich,目录号:NO202-146-2)(参见Recipes)
  18. 乙酸铀(Art,目录号:8473)(参见配方)
  19. 柠檬酸铅(TAAB,目录号:L003)(参见配方)

设备

  1. Eppendorf管
  2. 离心机
  3. 真空干燥箱
  4. 金刚石刀(Diatome)
  5. Ultracut(Leica)
  6. TEM(HITACHI,型号:H-7100)

程序

注意:在这个过程前一天,将所有的滴管和注射器放入60℃的烘箱中,以除去所有填充物中的所有水。

  1. 处理后,用0.5%胰蛋白酶-EDTA收集细胞(1×10 6),并通过离心机(300×g离心管)在eppendorf管中用0.5ml 0.1M磷酸盐缓冲液(PB)/em,5分钟)。
  2. 加入0.5毫升多聚甲醛(4%,在PB中)将细胞在室温(RT)下固定至少30分钟(或储存细胞在4℃长期储存)。
  3. 通过离心机(300×g,5分钟)用0.5ml 0.1M PB洗涤细胞3次。
  4. 加入0.5毫升四氧化四锇(1%在PB中)到管中,并在室温下孵育细胞1小时。
  5. 通过离心机(300×g,5分钟)用0.5ml 0.1M PB洗涤细胞3次。
  6. 加入0.5ml 70%酒精5分钟两次,离心(300×g,5分钟),脱水细胞→除去酒精。
  7. 加入0.5ml 85%酒精5分钟两次→离心(300×g,5分钟)脱水细胞→除去酒精。
  8. 加入0.5ml 95%酒精5分钟,三次离心细胞→离心(300×g,5分钟)→除去酒精。
  9. 脱水细胞,加入0.5ml 100%酒精10分钟,5次→离心(300×g,5分钟)→除去酒精。
    注意:从第10步开始,所有材料都应使用60°C烤箱。
  10. 将0.5ml环氧丙烷加入管中,并在RT下温育3分钟两次→离心(300×g,5分钟)→除去环氧丙烷。
  11. 向管中加入1ml(环氧丙烷:Epon = 1:1)并在室温下孵育1小时→离心(300×g,5分钟)→除去环氧丙烷:Epon。
  12. 将1ml纯Epon加入管中,并将样品放入真空干燥炉中,在室温下真空1小时。→除去Epon。
  13. 将1ml纯Epon加入管中,在室温下真空1小时。→除去Epon
  14. 将1ml纯Epon加入试管中,在室温下真空过夜
  15. 将管放入60℃烘箱至少48小时
  16. 修剪 - 移除块的边缘。
  17. 使用金刚石刀获得厚切片(1μm)Ultracut→将厚切片放在载玻片上,用0.5%甲苯胺蓝染色约40秒至1分钟(直到边缘干燥)→用水洗涤1分钟→观察 在显微镜下找到细胞
  18. 一旦我们发现细胞在厚的部分,改变Ultracut的厚度得到薄部分(65-70 nm)。
  19. 将薄片放在网格上(一个网格上3-4个部分)。
  20. 用醋酸铀和柠檬酸铅依次染色3分钟
  21. 用水清洗网格1分钟,让它们完全干燥
  22. 从相同壮观的TEM上的数码相机获取图像。

食谱

  1. 0.1M磷酸盐缓冲液(PB)
    19ml 0.2M NaH 2 PO 4 4/v/v 81ml 0.2M Na 2 HPO 4水溶液
  2. 4%多聚甲醛
    4%多聚甲醛在PB中的溶液
  3. 四氧化锇
    1%的四氧化锇在PB中
  4. Epon
    使用注射器混合以下所有试剂以制造Epon Poly/Bed 812 10 ml
    Araldite 502 6 ml
    DDSA 18ml
    DMP-30 0.7ml
    Epon可以在4°C或-20°C下储存1-2周
  5. 甲苯胺蓝
    0.5%甲苯胺蓝
    1%硼酸钠/H 2 O 2 / 用0.22μM过滤器过滤
  6. 乙酸乙烯酯
    在50%的乙醇中的饱和乙酸铀酰
  7. 柠檬酸铅
    1%的H 2 O中的柠檬酸铅 加入3滴10M NaOH(约0.5ml)

致谢

该协议改编自Chau和Lu(1996)和Lee等人(2012)。

参考文献

  1. Chau,Y.P。和Lu,K.S.(1996)。 在啮齿类动物的各种交感神经节中血微血管系统的差异渗透性。 Anat胚胎 (Berl) 194(3):259-269。
  2. Lee,C.P.,Liu,P.T.,Kung,H.N.,Su,M.T.,Chua,H.H.,Chang,Y.H.,Chang,C.W.,Tsai,C.H.,Liu,F.T.and Chen, ESCRT机制由病毒性BFRF1蛋白募集至细胞核相关膜, em> Epstein-Barr病毒。 8(9):e1002904。
  • English
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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: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Peng, W., Lu, K., Lai, S., Shy, H. and Kung, H. (2013). Transmission Electron Microscopy (TEM) Protocol: Observation Details within Cells. Bio-protocol 3(13): e816. DOI: 10.21769/BioProtoc.816.
  2. Lee, C. P., Liu, P. T., Kung, H. N., Su, M. T., Chua, H. H., Chang, Y. H., Chang, C. W., Tsai, C. H., Liu, F. T. and Chen, M. R. (2012). The ESCRT machinery is recruited by the viral BFRF1 protein to the nucleus-associated membrane for the maturation of Epstein-Barr Virus. PLoS Pathog 8(9): e1002904.
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