Manual dissection and fixation of Drosophila egg chambers for whole-mount FISH

This article describes a procedure for the manual dissection and fixation of Drosophila egg chambers for whole-mount fluorescent in situ hybridization (FISH). Egg chambers are more challenging to fix than embryos. They are easily overfixed so that probes will not penetrate well into the oocyte nucleus, even if some hybridization is detected in the follicle and nurse cells closer to the surface. If late-stage oocytes are to be examined, the tissue must also be protected against osmotic shock before and during fixation, because this will activate metaphase-arrested nuclei to undergo the metaphase-anaphase transition. Warming the buffered formaldehyde to 30°C immediately prior to fixation tends to enhance probe penetration. In contrast to reported results with immunostaining, FISH is equally successful whether fixation is performed with freshly prepared formaldehyde or commercial formaldehyde solutions. Manual dissection of fixed ovarioles gives a much higher yield per adult than the blender technique (facilitating analysis of mutants), and younger egg chambers in particular will be enriched, although the procedure itself is more laborious.