Uranyless

Mice were anesthetized using isoflurane before decapitation and optic nerve extraction. Optic nerves were immediately transferred to a fixative solution (4% PFA, 2.5% glutaraldehyde in phosphate buffer with 0.5% NaCl, pH 7.4) and fixed overnight at 4 °C. Tissue preparation and EM were carried out as previously described^{111 (link)}. Briefly, following postfixation with 2% OsO₄ (Science Services) in 0.1 M phosphate buffer (pH 7.3) and acetone dehydration, tissue fragments were embedded in EPON (Serva). Ultrathin sections were cut with a Leica UC7 ultramicrotome (Leica) and then stained using UranyLess (Science Services). EM pictures were captured with a Zeiss EM912 electron microscope (Zeiss) equipped with an on-axis 2k charge-coupled device camera (TRS). EM image analysis was performed using ImageJ (Fiji, version 1.52p). Axonal diameters and g ratios (ratio of axon diameter to the diameter including the myelin sheath) were analyzed using five random overview EM pictures (at 8,000× magnification), with 250 axons evaluated per animal. Calculations were based on circular areas equivalent to the measured areas. For axonal pathology assessment, eight to ten EM images per animal were analyzed. The experimenters were blinded to the genotypes.

E. coli suspensions at 5 × 10⁸ CFU/ml were mixed with peptides at the corresponding MICs and 0.5× MICs in PBS, and incubated for 1 hr at 37°C (incubator Thermomixer C, Eppendorf, Germany). Control samples (no AMPs) were suspended and incubated in PBS. Cell suspensions were centrifuged at 1300 × $g$ for 4 min in 1.5 ml Eppendorf tubes and resuspended (fixed) in 3% vol/vol glutaraldehyde and 0.1 M cacodylate buffer to bring the rapid cessation of biological activity and to preserve the structure of the cell. After fixation, the samples were washed and postfixed in 1% vol/vol OsO₄ in 0.1 M cacodylate buffer. Dehydration was carried out in an ascending ethanol series followed by two steps with propylene oxide (Hayat, 1989 (link)) and embedded in Agar Low Viscosity Resin (Agar Scientific, Stansted, UK). Ultrathin sections (70–80 nm) were prepared on an Ultramicrotome UC6 (Leica Microsystems, Vienna, Austria) equipped with a 35° Ultra Diamondknife (Diatome, Nidau, Switzerland). The grids were poststained with Uranyless (Science Services, Munich, Germany) and lead citrate according to Reynolds (Hayat, 1989 (link)). TEM images were acquired with Tecnai T12 at 120 kV (TFS, Warmond, Netherlands).

Open book preparations of third instar larvae were fixed in 4% PFA and 0.5% GA for 10 min, followed by 1 h fixation in 2% GA in 0.1 m sodium cacodylate buffer at room temperature, and a washing step in cacodylate buffer for one hour at room temperature. The larvae were postfixed on ice in the dark with 1% OsO₄ in 0.8% KFeCN, washed with NaCac buffer for 1 h on ice and stained with 1% uranyl acetate (UAc) on ice. Subsequently, the preparations were dehydrated in ethanol, followed by ethanol/epon solution and finally in pure liquid Epon overnight. Then the samples were embedded in a single drop of Epon. Muscle 6/7 of abdominal segments 2 to 5 were dissected, and multiple muscles were stacked and embedded in an Epon block, which was heated overnight at 60°C for polymerization. Ultrathin sections (60 nm) were prepared using a Leica UC7, mounted on copper electron microscopy grids and counter stained using Uranyless (Science services) and Reynolds lead citrate. The sections were examined at varying nominal magnifications using a 120 kV FEI Tecnai Spirit TEM microscope equipped with a F416 CMOS camera (TVIPS).