title: A simple approach for multicolor immunofluorescence staining in different Drosophila cell types.
abstract: Multicolor immunostaining analysis is often a desirable tool in cell biology for most researchers. Nonetheless, this is not an easy task and often not affordable by many laboratories as it might require expensive instrumentation and sophisticated analysis software. Here, we describe a simple protocol for performing sequential immunostainings on two different Drosophila specimens. Our strategy relies on an efficient and reproducible method for removal primary antibodies and/or fluorophore-conjugated secondary antibodies that does not affect antigene integrity. We show that alternation of multiple rounds of antibody incubation and removal on the same slide, followed by registration of the same DAPI-stained image, provides a simple framework for the sequential detection of several antigens in the same cell. Given that the sample fixation procedures used for Drosophila tissues are compatible with most specimen processing protocols, we can envisage that the multicolor immunostaining strategy presented here can be also adapted to different samples including mammalian tissues and/or cells.
authors.affiliation: Dipartimento di Biologia e Biotecnologie "C. Darwin", SAPIENZA Università di Roma, Roma, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cipressa
authors.forename: Maria Laura
authors.initials: ML
authors.lastname: Di Giorgio
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cenci
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000941:Antigens / Q000032:analysis*; D002843:Chromatin / Q000737:chemistry / Q000276:immunology; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis; D003116:Color; D004330:Drosophila / Q000166:cytology*; D029721:Drosophila Proteins / Q000032:analysis; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D006657:Histones / Q000032:analysis; D007211:Indoles; D007814:Larva / Q000166:cytology; D008297:Male; D059007:Polytene Chromosomes / Q000737:chemistry / Q000648:ultrastructure; D012469:Salivary Glands / Q000166:cytology; D013090:Spermatocytes / Q000166:cytology; D013194:Staining and Labeling / Q000379:methods*; D013737:Testis / Q000166:cytology; D044763:Ubiquitin-Conjugating Enzymes / Q000032:analysis
chemical_list: D000906:Antibodies; D000941:Antigens; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D029721:Drosophila Proteins; D005456:Fluorescent Dyes; D006657:Histones; D007211:Indoles; C471206:cav protein, Drosophila; C506595:heterochromatin protein 2, Drosophila; D000090266:Chromobox Protein Homolog 5; C007293:DAPI; D044763:Ubiquitin-Conjugating Enzymes; C471611:eff protein, Drosophila