Imaging Immunolabeled Drosophila Embryos by Confocal Microscopy

Monoclonal and polyclonal antisera raised against recombinant proteins are highly sensitive probes that reveal cellular and subcellular protein localization in developing embryos. For Drosophila researchers, the ease of generating such antisera (1 ) and the number and widespread availability of existing antibodies make immunofluroescence of embryos an indispensable technique. The use of fluorochrome-conjugated secondary and/or tertiary antibodies on Drosophila embryos and detection by confocal microscopy offers two, critical advantages over enzyme-mediated detection methods, such as alkaline phosphatase and horseradish peroxidase. First, the sensitivity achieved with confocal microscopy may be difficult to match with enzyme-mediated detection especially when imaging cells deep within later stage embryos. Second, immunofluorescence and confocal detection allow the selective and simultaneous labeling with up to three different primary antisera. The following protocol contains instructions for the fixation, labeling, and detection of Drosophila embryos with one, two, and three different primary antisera.