Jem arm 1300s high voltage electron microscope

After RAS FLS were washed five times with 0.1 M cacodylate buffer containing 0.1% CaCl₂ at 4°C, cells were fixed with 1% OsO₄ in 0.1M cacodylate buffer (pH 7.2) containing 0.1% CaCl₂ for 1 h at 4°C. After rinsing with cold distilled water, cells were dehydrated slowly with an ethanol series and propylene oxide at 4°C. The samples were embedded in EMbed-812 (EMS, 14120). After polymerization of the resin at 60°C for 36 h, serial sections were cut with a diamond knife on an LEICA EM UC6 ultramicrotome (Leica) and mounted on formvar-coated slot grids. Sections were stained with 4% uranyl acetate and lead citrate, and examined under a Tecnai G2 Spirit Twin transmission electron microscope (FEI Company) and a JEM ARM 1300S high voltage electron microscope (JEOL).

For transmission electron microscopy analysis, BMDMs were washed with PBS, fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h, post-fixed in 1% osmium tetroxide and 0.5% potassium ferricyanide in cacodylate buffer for 1 h, embedded in resin and cured at 80 °C for 24 h. Ultrathin sections (70–80 nm) were cut using an ultramicrotome (RMC MT6000-XL), stained with uranyl acetate and lead citrate, and examined using a Tecnai G2 Spirit Twin transmission electron microscope (FEI Co.) and a JEM ARM 1300S high-voltage electron microscope (JEOL, Tokyo, Japan).

For transmission electron microscopy analysis, infected lung tissues or BMDMs were washed with PBS, fixed with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h, post-fixed in 1% osmium tetroxide and 0.5% potassium ferricyanide in cacodylate buffer for 1 h, embedded in resin, and cured at 80 °C for 24 h. Ultrathin sections (70–80 nm) were cut using an ultramicrotome (RMC MT6000-XL), stained with uranyl acetate and lead citrate, and examined using a Tecnai G2 Spirit Twin transmission electron microscope (FEI Co., Hillsboro, OR, USA) and a JEM ARM 1300 S high-voltage electron microscope (JEOL, Tokyo, Japan).