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果蝇幼虫唾液腺组织中线粒体荧光蛋白标记观察   

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摘要:线粒体是细胞内重要的能量与物质代谢中心。可以通过免疫荧光染色法和线粒体荧光蛋白 (LaJeunesse等,2004) 标记法观察并检测细胞内线粒体的形态、大小与分布状态。本实验通过线粒体定位的EYFP来观察果蝇组织细胞中线粒体的形态大小与分布状态。

关键词: 果蝇, 唾液腺, 脂肪组织, 线粒体, 线粒体荧光标记蛋白

材料与试剂

  1. 培养皿
  2. 载玻片和盖玻片
  3. (可选) 过滤灭菌器
  4. EP管
  5. 锡箔纸
  6. 果蝇样本
  7. 甘油
  8. 指甲油
  9. NaCl
  10. KCl
  11. Na2HPO4
  12. KH2PO4
  13. HCl
  14. 多聚甲醛
  15. 1× PBS缓冲液 (见溶液配方)
  16. 4% PFA溶液 (见溶液配方)
  17. DAPI母液 (20 ng/μl) (见溶液配方)
  18. 0.1 M磷酸钠缓冲液 (见溶液配方)

仪器设备

  1. 解剖镊
  2. (可选) 高压蒸汽灭菌锅 
  3. -20 °C冰箱
  4. 激光共聚焦显微镜或者荧光显微镜 (Leica, model: Leica SP8)

实验步骤

  1. 将带有sqh-EYFP-Mito的果蝇亲本 (BL7194) 与待检测的果蝇亲本杂交;幼虫在实验条件下饲养到三龄幼虫Wandering时期。
    注:若用UAS-GFP,购买BDSC果蝇编号为BL8442。实验条件温度若无GAL4系统则用25 °C培养,若用UAS-GAL4系统,则用29 °C培养。
  2. 在PBS缓冲液中用解剖镊解剖三龄幼虫分离唾液腺组织;将组织放置于含有PBS缓冲液的染色皿中。
  3. 吸干PBS缓冲液,用4%多聚甲醛 (PFA) 在室温下固定30 min。
  4. 吸干PFA溶液,用PBS清洗3次。每次10 min。
  5. 按照1:10稀释20 ng/μl DAPI母液,并染色5 min。
  6. 吸走DAPI,用PBS清洗3次。
  7. 80%甘油压片,用指甲油封片。
  8. 用激光共聚焦显微镜或者荧光显微镜扫描样品 (图1)。检测光谱为EYFP检测光谱,激发光488 nm波长。


    图1. 果蝇唾液腺细胞mito-EYFP

溶液配方

  1. 1× PBS缓冲液
    137 mM NaCl
    2.7 mM KCl
    10 mM Na2HPO4
    2 mM KH2PO4
    用800 ml蒸馏水溶解8 g NaCl,0.2 g KCl, 1.44 g Na2HPO4 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. 4% PFA
    10 g多聚甲醛溶解在0.1 M磷酸钠缓冲液 (250 ml),加热60 °C搅拌至澄清,调整pH到7.4
  3. DAPI母液
    1 mg/ml
    10 mg DAPI粉末加入100 ml双蒸水中配置成1 mg/ml母液,分装在10个EP管中,用锡箔纸包裹,置于-20 °C冰箱保存。实际使用时,将母液1:5,000稀释
  4. 0.1 M磷酸钠缓冲液
    3.0 g NaH2PO4, 14.2 g Na2HPO4,溶解到1,250 ml蒸馏水中

参考文献

  1. LaJeunesse, D. R., Buckner, S. M., Lake, J., Na, C., Pirt, A. and Fromson, K. (2004). Three new Drosophila markers of intracellular membranes. Biotechniques 36(5): 784-788, 790.
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:杨晓, 黄勋. (2019). 果蝇幼虫唾液腺组织中线粒体荧光蛋白标记观察. Bio-101: e1010285. DOI: 10.21769/BioProtoc.1010285.
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