Formvar coated copper grid

Laminae in adult flies were processed for TEM imaging as described [52 (link)]. Samples were processed using a Ted Pella Bio-Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25°C in 4% paraformaldehyde, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4°C for 48 hours. 1% osmium tetroxide was used for secondary fixation and subsequently dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Science, Hatfield, PA). 50 nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Science, Hatfield, PA). 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.

Following propagation, bacteriophages CAP1 and CAP3 were incubated for 5 min at room temperature with host strain LH6 (OD₆₀₀ = 0.5) at MOI of ~100. Similarly, 50 µL of concentrated SLAP1 was incubated for 2 min with 250 µL LH6 cells at OD₆₀₀ = 0.5. Then 5% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) was added to fix the samples and halt the infection. The samples were spotted onto parafilm and formvar-coated copper grids (Electron Microscopy Sciences) were placed on top of each drop and left to incubate for 1 h at room temperature, followed by three 3 min washes in each 1x PBS and ddH₂O. The grids were negatively stained with 0.5% phosphotungstic acid (Electron Microscopy Sciences) for 10–30 s, wicked onto a Kimwipe and imaged with the JEOL JEM1011 TEM (JEOL Inc., Akishima, Tokyo, Japan).

Samples were stained and visualized essentially as described previously [10 (link), 90 (link)]. Aliquots of freshly thawed protein samples were adsorbed (10 μl) neat onto formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA) for 1 min before 10 μl of 0.25% glutaraldehyde (ThermoFisher, Waltham, MA) was added and incubated for 1 min. Thereafter, grids were wicked dry using qualitative filter paper (VWR, Radnor, PA), washed twice with 10 μl MilliQ water and then stained with 1% uranyl acetate for 2 min. Grids were wicked dry as above and allowed to air dry for at least 10 min, stored at room temperature and then examined using a 1200EX microscope (JEOL).
For measurement of soluble protein aggregates, at least three grids per protein were mounted, and at least three images per protein were analyzed. Length, defined as the long axis of each fragment measured, was measured using FIJI [77 (link)] and binned into 8 nm segments [66 (link)] using the scale bar as a standard (2.58 pixels/nm). Particles were excluded from analysis if they were on the border of the image or did not have clearly defined edges (i.e. due to a clustering). All particles meeting these criteria within the image field of view were also analyzed for width, defined as the short axis of each measured fragment.

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.

Gravid adults were frozen substituted in acetone (J.T. Baker, Phillipsburg, NJ, USA) containing 2% osmium tetroxide (Electron Microscopy Science, Hatfield, PA, USA) and 0.2% uranyl acetate (Electron Microscopy Science) in a Leica EM automatic freeze substitution machine (Leica). The temperature was raised from −90 °C to room temperature for 22 h (5 °C/h). The samples were dehydrated in 100% ethanol (Sigma-Aldrich) for 24 h, followed by two washes. The washed samples were embedded in the EMBed-812 embedding kit (Electron Microscopy Science) and incubated at 70 °C for 48 h. The 70 nm thin sections were cut by using a Reichert ultracut S microtome (Leica). Thin sections were collected on Formvar-coated copper grids (Electron Microscopy Science) and post-stained with 0.8% uranyl acetate (Electron Microscopy Science) in 40% methanol (J.T.Baker) followed by aqueous lead citrate (Electron Microscopy Science). Thin sections were viewed in a JEM-1230 transmission electron microscope (JEOL, Akishima, Tokyo, Japan).

A drop of each NP suspended in ultrapure water was placed on 300-mesh Formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 min. Excess liquid was blotted with filter paper. Grids were imaged using a Morgagni 268 TEM (FEI, Netherlands) at an accelerating voltage of 60 kV.