Visualization of Germinosomes and the Inner Membrane in <em>Bacillus subtilis</em> Spores

Juan Wen; Raymond Pasman; Erik  M.M. Manders; Peter Setlow; Stanley Brul

doi:10.3791/59388

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

枯草芽孢杆菌中细菌体和内膜的可视化

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08:58 min

April 15, 2019

DOI: 10.3791/59388-v

Juan Wen¹, Raymond Pasman¹, Erik M.M. Manders*^2,3, Peter Setlow*⁴, Stanley Brul*¹

¹Molecular Biology and Microbial Food Safety, Swammerdam Institute for Life Sciences,University of Amsterdam, ²Van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences,University of Amsterdam, ³Confocal.nl BV., ⁴Dept. of Molecular Biology and Biophysics,UConn Health

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萌发细胞受体蛋白聚集在枯草芽孢杆菌内膜的 "生殖细胞" 中。我们描述了一个协议, 使用超级分辨率显微镜和荧光报告蛋白可视化生殖细胞。该协议还确定了优先与 fm4-64 的膜染料染色的孢子内膜域。

超分辨率结构化照明显微镜3D-SIM是一种创新技术，用于在孢子膜中对发芽受体簇的荧光测量器蛋白进行可视化。使用这些程序，可以实现细菌体定位和孢子内膜脂域的无与伦比的分辨率。这项技术将由博士生胡安·温和我们实验室的理学硕士学生雷蒙德·帕斯曼展示。

在开始手术之前，用一摩尔盐酸清洁高精度盖玻片，在轻轻摇动的水浴中清洁30分钟，随后在超纯1型水中清洗两到五分钟。第二次洗涤后，将盖玻片放在 100% 乙醇中约 3 分钟，然后干燥盖玻片并验证其清晰度。然后，用 70% 乙醇清洁玻璃显微镜滑动一分钟，然后让幻灯片干燥并验证其清晰度。

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