Histology of L4 aphid head-stylets

Aphids were fixed overnight in 4% paraformaldehyde (PFA)-PBS solution at 4°C. After fixation, the samples were washed 3 times for 10 min each in phosphate buffered saline (PBS). The aphids were then transferred into aqueous 35% H2O2 for one week until the cuticle turned light-yellow. After washing again 3 times for 10 min each in PBS, the samples were incubated in graded ethanol solutions (50%, 70%, 90%, 95% and 100%; 10 min each). The samples were finally transferred to methyl salicylate (M6752, Sigma-Aldrich) for clearing. .tif stacks are opened with any bitmap image processing software (ImageJ). .ims file is open data file format (Bitplane Inc), and may be treated with Imaris (5.x File Format) or converted with ImarisFileConverter. Both accessible at biplane.com or https://imaris.oxinst.com/packages.