Multicolor fluorescence imaging of whole-mount Drosophila testes for studying spermatogenesis
Multicolor fluorescence imaging of whole-mount Drosophila testes for studying spermatogenesis
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DOI：
10.1016/j.ab.2013.01.009
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发表时间：
2013-05-01
影响因子：
2.9
通讯作者：
Olenina, Ludmila V.
中科院分区：
文献类型：
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作者：
Kibanov, Mikhail V.;Kotov, Alexei A.;Olenina, Ludmila V.
果蝇睾丸通常被认为是研究异性配子生物基本发育过程的有用模型。然而，整个果蝇睾丸的免疫染色通常与亚细胞水平的分辨率不足、重复性差以及固定标本的染色不完整有关。适当染色的主要问题是器官对抗体和抗体偶联荧光团的渗透性很差。为了克服这个问题，我们开发了一种完整的睾丸免疫染色方案，为共聚焦显微镜提供高质量的准备。许多亚细胞结构可以被成功地分辨出来，如光谱体、梭形体体、核颗粒、凋亡体和蛋白质晶体。这种方法保留了睾丸的内部结构，能够从一组共焦切片重建3D图像。它允许人们将荧光标记和免疫染色的蛋白质的同时检测与TUNEL分析相结合，以检测细胞凋亡。(C)2013 Elsevier Inc.保留所有权利。
Drosophila testes are generally considered a useful model for studying the fundamental developmental processes of heterogametic organisms. However, immunostaining of the whole Drosophila testis is often associated with insufficient resolution at the subcellular level, poor reproducibility, and incomplete staining of fixed preparations. The main problem for adequate staining is poor permeability of the organs for antibodies and antibody-coupled fluorophores. To overcome this problem we developed a protocol for whole-mount testis immunostaining yielding high-quality preparations for confocal microscopy. Many subcellular structures can be successfully resolved, such as the spectrosome, fusome, nuage granules, apoptotic bodies, and protein crystals. This method preserves the inner architecture of the testes, enabling 3D image reconstruction from a set of confocal sections. It allows one to combine the simultaneous detection of fluorescently tagged and immunostained proteins as well as TUNEL analysis for apoptosis detection. (C) 2013 Elsevier Inc. All rights reserved.