Original research article

A brief version of this protocol appeared in:
Rice Protocol e-book

Navigate this Article


果蝇脂肪组织高压冷冻-冷冻替代透射电镜 (HPF-FS TEM)   

How to cite Favorites Q&A Share your feedback Cited by


关键词: 高压冷冻固定, 透射电镜, 果蝇, 脂肪组织


  1. 果蝇三龄幼虫
  2. 液氮
  3. 锇酸 (四氧化锇)
  4. 丙酮
  5. 醋酸双氧铀
  6. Embed 812树脂
  7. NaCl
  8. KCl
  9. Na2HPO4
  10. KH2PO4
  11. HCl
  12. 1× PBS (见溶液配方)
  13. 样品替代液 (见溶液配方)


  1. Leica Microsystems HPM 100高压冷冻仪
  2. Leica Microsystems AFS-2冷冻替代仪
  3. Leica Ultracut UCT切片机
  4. JEM-1400 TEM电子显微镜
  5. Gatan832 4k × 2.7k CCD
  6. 烘箱


  1. 在1× PBS中解剖出来的三龄幼虫脂肪体进入Leica Microsystems HPM 100高压冷冻固定,并自动进入液氮。
  2. 样品在液氮中被转移到替代液中并置入Leica Microsystems AFS-2冷冻替代仪 -90 °C孵育72小时。
  3. 以8 °C每小时的速率升温到-60 °C并在此温度维持12小时。
  4. 再次以5 °C每小时的速率升温到-30 °C并在此温度维持10小时。
  5. 再升温至10 °C并维持10小时。
  6. 用纯的丙酮将样品快速洗四次。然后用1%的醋酸双氧铀染色1小时,然后用纯丙酮洗三次。
  7. 用Embed 812树脂梯度渗透,按丙酮对树脂比例2:1渗透3小时,1:1渗透5小时。然后用纯树脂渗透两次,每次8小时。
  8. 将样品转移至新鲜纯树脂中,60 °C烘箱中包埋聚合3天。
  9. 用Leica Ultracut UCT切片机进行超薄 (60 nm) 切片,并将切片收集排列于槽铜网格。
  10. 用JEM-1400 TEM电子显微镜在80 kV下操作观察,用Gatan832 4k × 2.7k CCD采集图片 (Ding等,2018)。


  1. 1× PBS
    137 mmol/L NaCl
    2.7 mmol/L KCl
    10 mmol/L Na2HPO4
    2 mmol/L KH2PO4
    用800 ml蒸馏水溶解8 g NaCl,0.2 g KCl, 1.44 g Na2PHO4和0.24 g KH2PO4
    用HCl调节溶液的pH值至7.4,加水至1 L
    分装后在15 psi (1.05 kg/cm2) 高压蒸汽灭菌20 min,或通过过滤除菌,保存至室温
  2. 样品替代液


  1. Ding, L., Yang, X., Tian, H., Liang, J., Zhang, F., Wang, G., Wang, Y., Ding, M., Shui, G. and Huang, X. (2018). Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism. EMBO J 37(17).
Please login or register for free to view full text
Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:丁隆, 黄勋. (2019). 果蝇脂肪组织高压冷冻-冷冻替代透射电镜 (HPF-FS TEM). Bio-101: e1010302. DOI: 10.21769/BioProtoc.1010302.
How to cite: Ding, L. and Huang, X. (2019). High Pressure Freezing-freezing Substitution Transmission Electron Microscope (HPF-FS TEM) of Drosophila Fat Tissue. Bio-101: e1010302. DOI: 10.21769/BioProtoc.1010302.

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.