Intracellular dye injection of previously immunolabeled insect neurons in fixed brain slices

Our method combines intracellular dye injection and immunohistochemistry. Under optical control, Lucifer Yellow was injected into immunohistochemically identified neurons that reside in fixed tissue. The technique allows visualization of the complete arborization patterns of immunostained neurons. Injections were performed on small neurons (somata < 10 microns in diameter). The technique works on microslices of insect brain. Standard immunohistochemical procedures have only been varied slightly, omitting Triton X-100 treatment. Anti-Lucifer Yellow immunohistochemistry, or alternatively the photoconversion technique, enables extension of the morphological analysis of these cells to the electron microscopic level. In the present study, Lucifer Yellow injections were performed on immunohistochemically pretreated brain microslices (anti-Locusta tachykinin II antiserum) of the beetle Tenebrio molitor.