Em 208 electron microscope

The diseased M. javanica J2 filled with BN-LEV cells were extracted from soil as previously described and selected for TEM preparation. The nematodes were handpicked from the suspension obtained by sieving and examined in temporary water mounts with LM at 250–400× to check occurrence of the disease. The diseased specimens were then embedded in 2% agarose, fixed in the glutaraldehyde–cacodylate buffer for 3 h or overnight, post-fixed in 1% OsO₄ in the cacodylate buffer, dehydrated in an ethanol series with 0.5% uranyl acetate at 70% ethanol, and then washed three times in 100% ethanol and propylene oxide before embedding the blocks in the Polybed resin. Serial sections 60–80 nm thick were cut with a Reichert microtome, stained with uranyl acetate and lead citrate solutions, and examined with a Philips EM 208 electron microscope at 100 kV.

For immunogold electron microscopy, HCT116 cells were fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer for 30 minutes at room temperature. Cells were scraped from the dishes, transferred to an Eppendorf tube, and centrifuged for 1 minute in a microfuge to obtain cell pellets. The pellets were washed with 0.1 M cacodylate buffer, dehydrated in increasing concentrations of methanol at -20°C, embedded in Lowicryl K4M at -20°C, and polymerized with ultraviolet irradiation. Ultrathin sections were mounted on nickel grids and sequentially incubated with 0.1 M glycine in PBS for 15 minutes, 5% BSA in PBS for 1 hour, and the rabbit polyclonal anti-SUMO-1 antibody (FL-101, sc-9060, Santa Cruz Biotechnology) diluted 1:50 in PBS containing 1% BSA and 0.1 M glycine for 1 hour. After washing, the sections were incubated with goat anti-rabbit IgG coupled to 10-nm gold particles (diluted 1:50 in PBS containing 1% BSA; BioCell, UK). After immunogold labeling, the grids were stained with lead citrate and uranyl acetate and examined with a Philips EM208 electron microscope operated at 60 kV. For control, ultrathin sections were treated as described above, but without the primary antibodies.

Transmission electron microscopy experiments were performed on POD4. Kidney slices were washed twice with PBS and fixed in 2.5% cacodylate-buffered glutaraldehyde (pH 7.35). Following postfixation in phosphate-buffered 1.3% osmium tetroxide, tissues were rinsed in buffer, dehydrated in graded alcohol, and processed into polymerised blocks of epoxy resin. Sections for initial light microscopy to determine tissue quality and architecture and suitability for ultrastructure analyses were cut at 0.5 µm and stained with toluidine blue. For electron microscopy, thin sections were cut with a diamond knife, mounted on copper grids, stained with alkaline lead citrate and 8% uranyl acetate, and examined using a Philips EM 208 electron microscope (Philips, Eindhoven, The Netherlands) operating at 80 kV. Photography was performed with a Morada digital camera (Olympus SIS, Münster, Germany).