Hochapfel, Florian and Denk, Lucia and Maassen, Christine and Zaytseva, Yulia and Rachel, Reinhard and Witzgall, Ralph and Krahn, Michael P. (2018) Electron microscopy of Drosophila garland cell nephrocytes: Optimal preparation, immunostaining and STEM tomography. JOURNAL OF CELLULAR BIOCHEMISTRY, 119 (10). pp. 8011-8021. ISSN 0730-2312, 1097-4644
Full text not available from this repository. (Request a copy)Abstract
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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | NEPHROTIC SYNDROME; FUNCTIONAL-ANALYSIS; MUTATIONS; ULTRASTRUCTURE; ENDOCYTOSIS; DYSFUNCTION; HEMOLYMPH; REVEALS; SYSTEM; MODEL; endocytosis; high pressure freezing; nephrocyte; slit diaphragm; STEM; tomography; transmission electron microscopy |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Biology, Preclinical Medicine > Institut für Anatomie > Lehrstuhl für Molekulare und zelluläre Anatomie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 10 Oct 2019 10:10 |
| Last Modified: | 10 Oct 2019 10:10 |
| URI: | https://pred.uni-regensburg.de/id/eprint/13644 |
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