INTRODUCTIONThe fruit fly Drosophila melanogaster has long been used to study the genetic factors involved in development, and the ability to localize molecules within the organism that allow genetic manipulation can be quite useful. This article discusses some of the issues relating to fixation of various Drosophila tissues for analysis by immunofluorescence microscopy. References to specific fixation protocols are included. The proper fixation protocol will depend on the structure to be visualized, the degree of preservation required, the preservation of antigenicity of the molecules of interest, and the level of resolution of the subsequent imaging. In addition, the fixation of thick tissues requires a protocol that effectively fixes the interior of the sample while not cross-linking the matrix so heavily that antibodies or other probes cannot penetrate efficiently and be washed out of the tissue. Particular attention is given to the handling of embryos, because these are used frequently and require special consideration.