Electron microscopy of Drosophila garland cell nephrocytes: Optimal preparation, immunostaining and STEM tomography
| Autor: | Michael P. Krahn, Reinhard Rachel, Ralph Witzgall, Yulia Zaytseva, Florian Hochapfel, Lucia Denk, Christine Maaßen |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Microscopy Electron Scanning Transmission Chemistry Podocytes Cell Biology Biochemistry Fusion protein Cell biology law.invention 03 medical and health sciences Immunolabeling 030104 developmental biology Freeze substitution law Scanning transmission electron microscopy Slit diaphragm Ultrastructure Animals Drosophila Proteins Drosophila Electron microscope Molecular Biology Immunostaining |
| Zdroj: | Journal of cellular biochemistry. 119(10) |
| ISSN: | 1097-4644 |
| Popis: | Due to its structural and molecular similarities to mammalian podocytes, the Drosophila nephrocyte emerged as a model system to study podocyte development and associated diseases. Similar to podocytes, nephrocytes establish a slit diaphragm between foot process-like structures in order to filter the hemolymph. One major obstacle in nephrocyte research is the distinct visualization of this subcellular structure to assess its integrity. Therefore, we developed a specialized dissection and fixation protocol, including high pressure freezing and freeze substitution techniques, to improve the preservation of the intricate ultrastructural details necessary for electron microscopic assessment. By means of scanning transmission electron microscopy (STEM) tomography, a three-dimensional dataset was generated to further understand the complex architecture of the nephrocyte channel system. Moreover, a staining protocol for immunolabeling of ultrathin sections of Epon-embedded nephrocytes is discussed, which allows the reliable detection of GFP-tagged fusion proteins combined with superior sample preservation. Due to the growing number of available GFP-trap fly lines, this approach is widely applicable for high resolution localization studies in wild type and mutant nephrocytes. |
| Databáze: | OpenAIRE |
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