In recent years, Drosophila melanogaster has become an attractive model organism in
which to study the structure and development of the cellular immune components. The
emergence of immunological markers greatly accelerated the identification of the immune
cells (hemocytes), while the creation of genetic reporter constructs allowed unique
insight into the structural organization of hematopoietic tissues. However, investigation
of the hemocyte compartments by the means of immunological markers requires dissection
and fixation, which regularly disrupt the delicate structure and hamper the microanatomical
characterization. Moreover, the investigation of transgenic reporters alone can be
misleading as their expression often differs from the native expression pattern of
their respective genes. We describe here a method that combines the reporter constructs
and the immunological tools in live imaging, thereby allowing use of the array of
available immunological markers while retaining the structural integrity of the hematopoietic
compartments. The procedure allows the reversible immobilization of Drosophila larvae
for high-resolution confocal imaging and the time-lapse video analysis of in vivo
reporters. When combined with our antibody injection-based in situ immunostaining
assay, the resulting double labeling of the hemocyte compartments can provide new
information on the microanatomy and functional properties of the hematopoietic tissues
in an intact state. Although this method was developed to study the immune system
of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological
markers could become a versatile technique for in vivo studies in other biological
systems too.
