Bio wave microwave oven

Laminas of adult flies chronically exposed to 0.2 ppm spinosad for 20 days (controls exposed to equivalent volume of DMSO) were processed for TEM imaging as described (Luo et al., 2017 (link)). TEM of laminas of 20-day-old Canton-S and Canton-S Dα6 KO mutants aged in the absence of spinosad was also investigated. Samples were processed using a Ted Pella Bio Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25°C in 4% PFA, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4°C for 48 hr. 1% osmium tetroxide was used for secondary fixation with subsequent dehydration in ethanol and propylene oxide. Samples were then embedded in Embed-812 resin (Electron Microscopy Sciences, Hatfield, PA). 50-nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Sciences). Specimens were stained with 1% uranyl acetate and 2.5% lead citrate and imaged using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 megapixel CCD camera. For quantification of ultrastructural features, electron micrographs were examined from three different animals per treatment. The data were analyzed using Student’s unpaired t-test.

Only formalin fixed tissues were available for transmission electron microscopy (tEM). Tissues were re-fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. All processing was done with the assistance of a Pelco Biowave microwave oven (Ted Pella, Redding, USA). The samples were then washed with 0.1 M cacodylate buffer, and post fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer. They were further washed with ultra-high quality water then dehydrated through a graded acetone series and infiltrated with Epon resin and polymerised at 60°C for 2 days. Ultramicrotomy was performed on a Leica Ultracut UC6 ultramicrotome and sections of 50 nm thickness were mounted on copper grids and stained with 5% uranyl acetate in 50% methanol, followed by Reynolds lead citrate. The sections were viewed in a JEOL 1010 transmission electron microscope operated at 80 kV and images were collected on an Olympus Soft Imaging Veleta digital camera.