Slides were prepared as acetone fixed preparations of the purified antigens by experienced laboratory technicians capable of maintaining all conditions equal between test runs.
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.
Most previous studies have investigated these properties in fixed preparations, which make the distinction between regeneration and growth from spared fibres difficult, and can provide only minimal insights into key dynamic features.
We have followed mitotic spindle establishment by time-lapse microscopy and on fixed preparations.
For the smear test, fixed preparations were stained with auramine and visualized under a fluorescence microscope (at × 400 magnification).
Procedures used for imaging living or fixed preparations of cultured embryos are given in the Extended Experimental Procedures.
Moreover, the extra centrioles incorporated the centriole markers mRFP-Fzr or mRFP-PACT and, in fixed preparations, they recruited PCM markers (γ-tubulin, D-TACC, and Cnn) and organized microtubules (MTs) (not shown).
In general, imaging of these fine subcellular domains in live samples produced clearer and more consistent localization patterns than fixed preparations, leading us to use the live imaging approach whenever applicable, throughout the analysis.
The development of R7 axons, particularly with regards to their layer specificity (Ting, 2005; Lee, et al., 2001; Clandinin, et al., 2001) and columnar restriction (Ting et al., 2007; Ting, et al., 2014) has been studied extensively in fixed preparations.
