Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM).

摘要: Abstract Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing cuticle while preserving both, hard and soft tissues. The resulting transparent allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images e.g. brain, glands, muscles fine cuticular structures. Using fluorescent dye, even single labeled neurons can be visualized resolved up an imaging depth 150 μm through cleared Hydrogen-peroxide, was used clear cuticle, does preclude immunocytochemical techniques, shown successful labeling serotonin-immunoreactive (5HT-ir) in ants' brain. ‘transparent insect protocol’ presented here especially suited for small arthropods where dissection organs very demanding difficult achieve. Furthermore, preserved situ thus allowing more precise three-dimensional reconstruction compared to, e.g., dissected or sectioned tissue.