title: Immunofluorescent antibody staining of intact Drosophila larvae.
abstract: Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis. We have modified existing embryo protocols to develop a reliable method for antibody staining of whole Drosophila larvae of any developmental stage. The procedure consists of a bleach wash, enzymatic digestion, first fixation, 'cracking', second fixation, (optional) Proteinase K (Pro-K) or sonication treatment, antibody staining, clearing, and mounting. The method takes longer than typical antibody stains of dissected larval tissues-12 or 16 d, depending on the size of the larvae, compared with 2-3 d for embryos or dissected tissue stains-but time is saved by eliminating the need for larval dissections and by allowing hundreds of larvae to be batch-processed. The method also works well for staining embryos, even late-stage embryos with cuticles, allowing characterization from early embryogenesis to the end of larval development.
authors.affiliation: Institute of Molecular Biology, Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA.
authors.forename: Laurina
authors.initials: L
authors.lastname: Manning
authors.affiliation: Institute of Molecular Biology, Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, Oregon, USA.
authors.forename: Chris Q
authors.initials: CQ
authors.lastname: Doe
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000166:cytology / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods*; D007814:Larva / Q000166:cytology / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation
chemical_list: D019286:Endopeptidase K