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Processing of soft pupae and uneclosed pharate adults of Drosophila for scanning electron microscopy
Author(s) -
Beňo Milan,
Liszeková Denisa,
Farkaš Robert
Publication year - 2007
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20507
Subject(s) - osmium tetroxide , integument , fixative , scanning electron microscope , biology , anatomy , appendage , fixation (population genetics) , biomedical engineering , electron microscope , materials science , microbiology and biotechnology , biochemistry , composite material , medicine , physics , cytoplasm , gene , optics
For over four decades, scanning electron microscopy (SEM) has been used in research involving Drosophila genetics and developmental biology. It allows for observation and documentation of the gross morphology of exoskeletal structures as well as their characterization at very high resolution. In most cases, SEM in Drosophila has been limited to imaging adult heads, thoraces, appendages, and embryos, as these structures are relatively hard and/or easy to process for SEM. In contrast, the structures of the pharate adult stages are difficult to prepare for SEM because their integument is quite soft, they are extremely dirty and they are resistant to the usual processing methods. Here, we present an innovative method to prepare these types of structures. Our protocol efficiently removes extraneous material originating from the exuvial fluid of pharate adults and uses a hydrophobic expansion step to keep the soft exoskeleton of the body inflated. In addition to using immersion fixation, it utilizes fixation within the body that occurs via a reaction between osmium tetroxide and alcohols that are infiltrated into the body during a hydrophobic expansion step. This novel approach results in a properly inflated integument that retains its shape in subsequent procedures. Our method provides a useful, general alternative for processing difficult samples, including soft, biological “whole‐mount” specimens and samples that are extremely dirty or resistant to fixative penetration. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.