ABSTRACT
This protocol describes formaldehyde fixation of Drosophila testes. The procedure preserves chromosome morphology very well, allowing a clear visualization of the chromosome condensation-decondensation cycle. It also results in excellent preservation of microtubules for immunostaining. The main disadvantage of this fixation technique is its poor preservation of cell morphology, as viewed by phase-contrast optics. In addition, because it involves a rather hard squashing of formaldehyde-fixed testes, it results in meiotic figures which are flatter and larger than those obtained using methanol-acetone or paraformaldehyde fixation protocols.
Subject(s)
Drosophila/anatomy & histology , Entomology/methods , Tissue Fixation/methods , Animals , Fixatives/metabolism , Formaldehyde/metabolism , Male , Testis/anatomy & histologyABSTRACT
This protocol describes chromatin staining of Drosophila testes. To visualize DNA, preparations fixed using methanol-acetone, paraformaldehyde, or formaldehyde can be stained with several DNA-binding dyes. If the slides are to be examined with a fluorescence microscope equipped with filters that permit ultraviolet (UV) excitation, suitable dyes for DNA staining are Hoechst 33258 or 4',6-diamidino-2-phenylindole (DAPI). If the slides are to be analyzed with a confocal microscope not equipped with a UV laser, DNA can be stained with either propidium iodide or TOTO-3 iodide.
Subject(s)
Chromatin/metabolism , Coloring Agents/metabolism , Cytological Techniques/methods , Drosophila/cytology , Entomology/methods , Staining and Labeling/methods , Animals , Bisbenzimidazole/metabolism , DNA/metabolism , Indoles/metabolism , Male , Propidium/metabolism , Quinolines/metabolism , Testis/cytology , Thiazoles/metabolismABSTRACT
This protocol describes paraformaldehyde fixation of Drosophila testes. The procedure preserves F-actin-containing structures because it does not involve the use of methanol. Thus, it is particularly suitable for contractile ring and fusome visualization. However, it does not preserve cell morphology and microtubules as efficiently as methanol-acetone fixation and does not permit clear detection of the Y loops.
Subject(s)
Drosophila/anatomy & histology , Fixatives/metabolism , Formaldehyde/metabolism , Polymers/metabolism , Tissue Fixation/methods , Animals , Male , Testis/anatomy & histologyABSTRACT
Preparations of Drosophila testes fixed with paraformaldehyde can be stained for F-actin according to the protocol described here. This staining procedure is particularly suitable for staining the male fusome and the cytokinetic contractile ring.
Subject(s)
Actins/analysis , Drosophila/anatomy & histology , Staining and Labeling/methods , Animals , Fixatives/metabolism , Formaldehyde/metabolism , Male , Polymers/metabolism , Testis/anatomy & histology , Testis/chemistry , Tissue Fixation/methodsABSTRACT
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.
Subject(s)
Acetone/chemistry , Drosophila/metabolism , Methanol/chemistry , Testis/metabolism , Animals , MaleABSTRACT
Fixed Drosophila testis preparations can be immunostained with a variety of antibodies. This article describes a general procedure for immunostaining. The concentration of the primary antibody will vary with both the type of antibody and the type of incubation and should be determined empirically each time.