Multicolor fluorescence imaging of whole-mount Drosophila testes for studying spermatogenesis

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.