Methanol-acetone fixation of Drosophila testes

This protocol describes two techniques for methanol-acetone fixation of Drosophila melanogaster testis squashes. The first method results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics show most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation method results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is poor preservation of chromosome structure. In most instances, the chromosomes do not show a distinct morphology and tend to coalesce into one or more masses of chromatin. The second technique for methanol-acetone fixation described here has proved to be particularly suitable for γ-tubulin and centrosomin immunostaining. It results in preparations having the same characteristics as those obtained with the first method. © 2011 Cold Spring Harbor Laboratory Press.