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LC3B Labeling on Terrestrial Isopod Adipocytes
LC3B标记陆生等足目脂肪细胞   

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Abstract

The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes. To visualize the LC3B, we used the LC3B Antibody Kit for Autophagy (Invitrogen). As this kit was designed to work with cell coming from cell culture it was not possible to perform it on compact tissues such as nerve cord or ovaries. We thus adapted LC3B labeling initial protocol to be able to label autophagic membranes of adipocytes from terrestrial isopods which form a loose tissue that can be more easily penetrated by antibodies. The following protocol permits a visualization of the autophagic vesicles directly in terrestrial isopod cells freshly sampled in animals.

Materials and Reagents

  1. LC3B Antibody kit for autophagy (Life Technologies, Invitrogen™, catalog number: L10382 )
  2. Alexa fluor 555 goat anti-rabbit IgG (H + L) (2 mg/ml) (Life Technologies, Invitrogen™, catalog number: A21428 )
  3. Formaldehyde
  4. BSA
  5. DAPI (2.5 μg/ml) (Sigma-Aldrich)
  6. AF1 antifading-25 ml (Citifluor, England)
  7. Pincers for dissection
  8. Pap Pen Liquid Blocker Super Pap Pen (Daido Sangyo Co. Ltd, Japan)
  9. Glass slide
  10. Blocking buffer (see Recipes)
  11. Terrestrial isopod Phosphate buffered saline (PBS) (see Recipes)
  12. Fixative (see Recipes)
  13. Permeabilization reagent (see Recipes)

Equipment

  1. Black box
  2. Epifluorescent microscope (Axio Observer-A1, Zeiss) with Apotome (structured illumination) equipped with a 63x/1.25 objective (oil immersion) and with the AxioVision 4.8.1 software (Zeiss)

Procedure

  1. Dilute the LC3B rabbit polyclonal antibody in blocking buffer to prepare 0.5 μg/ml working solution (1 μl stock solution in 1,999 μl of blocking buffer).
  2. Draw a well (of a diameter of around 1 cm) with a liquid repellent pen on glass slide.
  3. Dissect the terrestrial isopod and collect fat tissue especially those surrounding the nerve cord (a length of around 6 mm) (Figure 1).

    Figure 1. Location of the nerve cord in Porcellio dilatatus (A) and the nerve cord surrounded by adipocytes (Ad) once collected (B).

  4. Deposit the nerve cord surrounded by adipocytes (fat tissue) on the middle of the drawn well.
  5. Add 150 μl of the 3.7% formaldehyde fixative in terrestrial isopod PBS onto the tissue.
  6. Incubate 15 min in a black box containing a humid paper at room temperature (RT).
  7. Remove the fixative and wash the tissue three times with terrestrial isopod PBS (5 min with 150 μl of PBS by washing).
  8. Remove the PBS after the last wash.
  9. Prepare 0.2% Triton X-100 in terrestrial isopod PBS and apply 150 onto the fat tissue.
  10. Be careful of not dissolving the liquid repellent pen for the tissue to be always immerged in the solution.
  11. Incubate in a black box with humid paper at 15 min RT.
  12. Draw a well (~1 cm diameter) with a liquid repellent pen on a fresh glass slide.
  13. Collect the fat tissue with forceps and transfer it onto the fresh slide.
  14. Add 150 μl of the diluted primary antibody prepared in step 1 onto the fat tissue.
  15. Incubate 60 min in a black box containing a humid paper at RT.
  16. Remove the primary antibody solution and wash the tissue three times with terrestrial isopod PBS (5 min with 150 μl of PBS by washing).
  17. Remove the PBS after the last washing.
  18. Dilute the goat anti-rabbit secondary antibody in blocking buffer to prepare 5 μg/ml working solution (1 μl stock solution in 399 μl of blocking buffer).
  19. Add 150 μl of goat anti-rabbit secondary antibody onto the fat tissue.
  20. Incubate 60 min in a black box with humid paper at RT.
  21. Remove the anti-goat secondary antibody solution and wash the cells three times with terrestrial isopod PBS (5 min with 150 μl of PBS by washing).
  22. Mount the preparation in a mixture of DAPI (2.5 mg/ml) to label the nuclei and Citifluor (AF1 antifading).
  23. Detection was performed with epifluorescent microscope with apotome equipped with a 63x/1.25 objective (oil immersion) and with the AxioVision 4.8.1 software (Figure 2).


    Figure 2. Observation of LC3B labeling in adipocytes collected from Porcellio dilatatus infected with the Wolbachia strain wVulC (Le Clec’h et al., 2012).

Recipes

  1. Terrestrial isopod PBS
    137 mM NaCl 
    1.5 mM Na2HPO4 
    8 mM KH2PO4 
    3 mM KCl 
    pH 7.4
  2. Blocking buffer: BSA 1% diluted in terrestrial isopod PBS 
  3. Fixative: 3.7% formadehyde in terrestrial isopod PBS
  4. Permeabilization reagent: 0.2% Triton X-100 in terrestrial isopod PBS

Acknowledgments

We thank all the technical staff of the UMR EBI CNRS 7267. We also thank Winka Le Clec’h for her valuable comments on the protocol. This work was supported by the Agence Nationale de la Recherche (ADaWOL ANR-09-JCJC-0109-01 coordinated by Mathieu Sicard).

References

  1. Chevalier, F., Herbiniere-Gaboreau, J., Bertaux, J., Raimond, M., Morel, F., Bouchon, D., Greve, P. and Braquart-Varnier, C. (2011). The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia. PLoS One 6(4): e18531.
  2. Le Clec'h, W., Braquart-Varnier, C., Raimond, M., Ferdy, J. B., Bouchon, D. and Sicard, M. (2012). High virulence of Wolbachia after host switching: when autophagy hurts. PLoS Pathog 8(8): e1002844.

简介

LC3B蛋白在自噬中起关键作用。 通常,这种蛋白质存在于细胞质中,但是在用磷脂酰乙醇胺切割和脂化后,LC3B与吞噬细胞结合。 这种定位可以用作自噬膜的一般标记。 为了显现LC3B,我们使用用于自噬的LC3B抗体试剂盒(Invitrogen)。 因为该试剂盒被设计用于来自细胞培养物的细胞,所以不可能在紧凑组织例如神经索或卵巢上进行。 因此,我们适应LC3B标签初始协议,以能够标记来自陆地isopods的脂肪细胞的自噬膜,形成松散的组织,可以更容易被抗体穿透。 以下方案允许直接在动物中新鲜取样的陆生等足细胞中观察自噬泡。

材料和试剂

  1. LC3B用于自噬的抗体试剂盒(Life Technologies,Invitrogen TM,目录号:L10382)
  2. Alexa fluor 555山羊抗兔IgG(H + L)(2mg/ml)(Life Technologies,Invitrogen TM,目录号:A21428)
  3. 甲醛
  4. BSA
  5. DAPI(2.5μg/ml)(Sigma-Aldrich)
  6. AF1抗褪色-25ml(Citifluor,England)
  7. 夹具用于解剖
  8. Pap Pen Liquid Blocker Super Pap Pen(Daido Sangyo Co.Ltd,Japan)
  9. 玻璃片
  10. 阻止缓冲区(参见配方)
  11. 陆地等度磷酸盐缓冲盐水(PBS)(参见配方)
  12. 固定剂(见配方)
  13. 渗透试剂(参见配方)

设备

  1. 黑框
  2. 使用配有63x/1.25物镜(油浸)和AxioVision 4.8.1软件(Zeiss)的Apotome(结构化照明)的荧光显微镜(Axio Observer-A1,Zeiss)

程序

  1. 在封闭缓冲液中稀释LC3B兔多克隆抗体以制备0.5μg/ml工作溶液(1μl储备溶液在1,999μl封闭缓冲液中)。
  2. 在玻璃载玻片上用防液笔画一口井(直径约1厘米)。
  3. 解剖地面同位素并收集脂肪组织,特别是那些围绕神经线(长度约6毫米)(图1)。

    图1.一旦收集(B),在肺泡肿瘤(A)和被脂肪细胞(Ad)包围的神经索的神经索的位置。

  4. 将被脂肪细胞包围的神经索(脂肪组织)沉积在所绘制的孔的中间。
  5. 加入150微升的3.7%甲醛固定剂在陆地等量PBS PBS组织
  6. 在室温(RT)下在含有湿纸的黑盒中孵育15分钟。
  7. 除去固定剂,用地面等离子体PBS(用洗涤150μlPBS洗5分钟)洗涤组织三次。
  8. 在最后一次清洗后取出PBS。
  9. 准备0.2%Triton X-100在陆地等度磷脂和施加150脂肪组织。
  10. 小心不要溶解拒液笔,使组织总是浸入溶液中。
  11. 在黑盒子中用湿纸在室温下15分钟孵育。
  12. 用防液笔在新鲜载玻片上绘制孔(〜1cm直径)。
  13. 用镊子收集脂肪组织,并将其转移到新鲜的幻灯片上。
  14. 加入150μl步骤1中制备的稀释的一抗在脂肪组织上。
  15. 在室温下在含有湿纸的黑盒中孵育60分钟。
  16. 取出一抗溶液,用地面等离子体PBS(用洗涤用150μlPBS洗5分钟)洗涤组织三次。
  17. 在最后一次洗涤后取出PBS。
  18. 在封闭缓冲液中稀释山羊抗兔二抗以制备5μg/ml工作溶液(1μl储备溶液,在399μl封闭缓冲液中)。
  19. 向脂肪组织中加入150μl山羊抗兔二抗。
  20. 在黑盒子中用湿纸在室温下孵育60分钟。
  21. 取出抗山羊第二抗体溶液,用地面等离子体PBS洗涤细胞三次(洗涤用150μlPBS洗涤5分钟)。
  22. 将制剂安装在DAPI(2.5mg/ml)的混合物中以标记细胞核和Citifluor(AF1抗褪色)。
  23. 用装有63x/1.25物镜(油浸)的开口机和AxioVision 4.8.1软件(图2)的落射荧光显微镜进行检测。


    图2.从用Wolbachia菌株wVulC感染的肺泡细胞收集的脂肪细胞中LC3B标记的观察(Le Clec'h 等 ,2012)。

食谱

  1. 陆地等度PBS
    137 mM NaCl 
    1.5mM Na 2 HPO 4。
    8mM KH 2 PO 4 4/
    3 mM KCl 
    pH 7.4
  2. 封锁缓冲区: BSA在地上等磷蛋白PBS中稀释1%
  3. 固定剂:3.7%甲醛在地上等量PBS中
  4. 渗透试剂:0.2%Triton X-100在地上等量PBS PBS中

致谢

我们感谢UMR EBI CNRS 7267的所有技术人员。我们还感谢Winka Le Clec'h对协议的宝贵意见。 这项工作得到了Agis Nationale de la Recherche(ADaWOL ANR-09-JCJC-0109-01由Mathieu Sicard协调)的支持。

参考文献

  1. Chevalier,F.,Herbiniere-Gaboreau,J.,Bertaux,J.,Raimond,M.,Morel, F.,Bouchon,D.,Greve,P。和Braquart-Varnier,C。(2011)。 陆地等足类动物免疫细胞效应物 Armadillidium vulgare :与其入侵者会面 ,Wolbachia。 PLoS One 6(4):e18531。
  2. Le Clec'h,W.,Braquart-Varnier,C.,Raimond,M.,Ferdy,J.B.,Bouchon,D。和Sicard,M。(2012)。 宿主切换后的Wolbachia高毒力:当自噬受到伤害时。 PLoS Pathog 8(8):e1002844。
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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. Braquart-Varnier, C., Raimond, M. and Sicard, M. (2013). LC3B Labeling on Terrestrial Isopod Adipocytes. Bio-protocol 3(12): e792. DOI: 10.21769/BioProtoc.792.
  2. Le Clec'h, W., Braquart-Varnier, C., Raimond, M., Ferdy, J. B., Bouchon, D. and Sicard, M. (2012). High virulence of Wolbachia after host switching: when autophagy hurts. PLoS Pathog 8(8): e1002844.
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