Park Karen Sophia, Godt Dorothea, Kalderon Daniel
Department of Biological Sciences, Columbia University.
Department of Cell and Systems Biology, University of Toronto.
J Vis Exp. 2018 Mar 2(133):56779. doi: 10.3791/56779.
Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.
与成年果蝇的卵巢不同,由于蛹期卵巢体积小、半透明且包裹在蛹壳内,相对难以获取和检查。解剖蛹期卵巢的挑战还在于它们在蛹体内的实际位置:卵巢被蛹腹部内的脂肪体细胞包围,必须去除这些脂肪细胞才能进行适当的抗体染色。为了克服这些挑战,本方案使用定制的巴斯德吸管从蛹腹部提取脂肪体细胞。此外,在染色过程中使用带腔盖玻片代替微量离心管,以提高蛹的可见度。然而,尽管本方案中使用的工具具有这些及其他优点,但由于蛹期卵巢体积小,成功执行这些技术可能仍需要几天的练习。本方案中概述的技术可应用于时间进程实验,在该实验中,在蛹发育的各个阶段对卵巢进行分析。