Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes
| Autor: | Roger E. Karess, Vincent Contremoulins, Natacha Raich |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
General Immunology and Microbiology
medicine.diagnostic_test Chemistry General Chemical Engineering General Neuroscience Confocal Colocalization Spermatocyte Immunofluorescence General Biochemistry Genetics and Molecular Biology law.invention Cell biology Immunolabeling medicine.anatomical_structure Confocal microscopy law medicine Nucleoporin Immunostaining |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes. |
| Databáze: | OpenAIRE |
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