Tecnai g2 spirit tem

Carbon film in 300-mesh grids (Agar Scientific) were glow discharged at 25 mA for 1 min. Samples were applied to the grids and washed with RNase-free water before staining with 2% (w/v) uranyl acetate. To visualize MDA5 filaments from the cooperativity assays, protein was incubated on ice for 30 min with 10 mM ATPγS with a final concentration of 200 nM MDA5 and 10 nM 1-kb dsRNA. To visualize LGP2-MDA5 complex formation, 73 nM MDA5 and 73, 146, or 730 nM LGP2 were incubated with 4 mM ATP and 4.4 nM 1-kb dsRNA on ice for 30 min. Samples containing MDA5 and 73, 146, and 730 nM LGP2 were rapidly diluted 1:2, 1:3, and 1:10 fold, respectively, before staining. All ns-EM images were collected on a 120 kV Tecnai G2 Spirit TEM (Thermo Fisher Scientific). Images were collected at −2 to −4 μm defocus, 26000× magnification, and 4 Å per pixel. Results were analyzed and filament length quantified with ImageJ (v1.5, imagej.net).

Spleen tissues were fixed with 2.5% glutaraldehyde solution overnight. Spleen tissues were then carefully washed with PBS (pH 7.4) thrice and postfixed with 1% osmium tetroxide for 1 h. Samples were washed with pure water thrice, stained with 2% uranium acetate for 30 min, dehydrated stepwise using 50%-100% ethanol and 100% acetone, permeated in resin and acetone mixture (1 : 1, v/v) at room temperature for 2 h, and subsequently embedded in resin. Embedded spleen specimens were cut into 70 nm ultrathin sections using Leica UC7 and viewed using a Tecnai G2 Spirit TEM (Thermo Fisher, CA, United States) operating at 80 kV.