Em 301 transmission electron microscope

Bacteriophages were concentrated and purified with Polyethylene Glycol (PEG) [20 ]. Briefly, DNase I and RNase were added to a flask containing 5 mL of each phage supernate to a final concentration of 1 µg/mL and incubated at 26 °C for 30 min. NaCl was added to a final concentration of 1 M and placed on ice or 1 h. In order to remove bacterial debris, samples were centrifuged at 11,000× g for 10 min at 4 °C. Maintaining a temperature of 4 °C, PEG 8000 was added to a final concentration of 10%. Tubes were rocked at 4 °C for 1.5 h. Tubes were centrifugated at 11,000× g for 10 min at 4 °C to precipitate the phage. The supernatant was carefully removed from the pellet and 1 mL of SM buffer was added to the pellet. Phage pellets were resuspended in 1 mL SM buffer and were stained with 2% aqueous (w/v) uranyl acetate adjusted to pH 4.2 and examined with a Philips EM 301 Transmission Electron Microscope operated at 60 kV. Bacteriophages were observed at high magnification (×71,000) [20 ]. The images were edited with ImageJ software version 1.46r.

The method of EM for third-instar larval NMJ6/7 was described in previous reports (44 (link), 46 (link)). Briefly, third-instar larvae were rapidly dissected in Ca²⁺ solution. The dissected body wall muscles (NMJ6/7; segment A3) were first fixed for 4 h at 4 °C in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 m sodium cacodylate buffer (pH 7.2) and then were fixed at 4% glutaraldehyde overnight. After being washed with washing buffer (0.1 m sodium cacodylate buffer, pH 7.2), the samples were post-fixed for 1.5 h with washing buffer containing 1% osmium tetroxide followed by staining for 1 h on ice with 1% uranyl acetate in distilled water. After being dehydrated at room temperature with increasing ethanol concentrations, the samples were infiltrated in Epon resin and embedded in three successive steps at 30, 45, and 60 °C, each lasting for 24 h. Series of 80–90-nm ultrathin sections were cut with a 35° diamond knife (Diatome) on a Reichert ultracut E ultramicrotome (Leica) and mounted on Formvar-coated grids. The sections were finally stained in uranyl acetate and lead citrate. Micrographs were obtained with a Philips EM 301 transmission electron microscope.