title: Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM).
abstract: Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the structures of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing of pigmented cuticle while preserving both, hard and soft tissues. The resulting transparent cuticle allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images of e.g. the brain, glands, muscles and fine cuticular structures. Using a fluorescent dye, even single labeled neurons can be visualized and resolved up to an imaging depth of 150 μm through the cleared cuticle. Hydrogen-peroxide, which was used to clear the cuticle, does not preclude immunocytochemical techniques, shown by successful labeling of serotonin-immunoreactive neurons (5HT-ir) in the ants' brain. The 'transparent insect protocol' presented here is especially suited for small arthropods where dissection of organs is very demanding and difficult to achieve. Furthermore, the insect organs are preserved in situ thus allowing a more precise three-dimensional reconstruction of the structures of interest compared to, e.g., dissected or sectioned tissue.
authors.affiliation: Department of Biology, Universitätsstraße 10, University of Konstanz, Konstanz 78457, Germany; Department of Behavioral Physiology and Sociobiology, Biozentrum, Am Hubland, University of Würzburg, Würzburg, Germany; Computational and Evolutionary Biology, Faculty of Life Science, University of Manchester, Manchester, United Kingdom. Electronic address: Marco.Smolla@postgrad.manchester.ac.uk.
authors.forename: Marco
authors.initials: M
authors.lastname: Smolla
authors.affiliation: Life Science Division, Max Schmidheiny-Strasse 201, Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland. Electronic address: Markus.Ruchty@leica-microsystems.com.
authors.forename: Markus
authors.initials: M
authors.lastname: Ruchty
authors.affiliation: Department of Biology, Universitätsstraße 10, University of Konstanz, Konstanz 78457, Germany; Department of Behavioral Physiology and Sociobiology, Biozentrum, Am Hubland, University of Würzburg, Würzburg, Germany. Electronic address: Manuel.Nagel@uni-konstanz.de.
authors.forename: Manuel
authors.initials: M
authors.lastname: Nagel
authors.affiliation: Department of Biology, Universitätsstraße 10, University of Konstanz, Konstanz 78457, Germany. Electronic address: Christoph.Kleineidam@uni-konstanz.de.
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Kleineidam
mesh_terms: D000818:Animals; D001000:Ants / Q000033:anatomy & histology*; D049628:Body Size; D021621:Imaging, Three-Dimensional / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*
keywords: Bleaching pigments; Hydrogen-peroxide; Immunocytochemistry; Insect anatomy; Serotonin; Transparent cuticle