A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy.

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton-derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four-step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate-based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline-based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point-dried, mounted on aluminum stubs, sputter coated with gold-palladium alloy and examined in the SEM.