Toluidine blue

RAW264.7 MΦ were infected as described above. Two hours after infection, cells were washed twice with PBS and harvested in PBS containing 2% gelatin (Sigma Aldrich, G9391-100 g). Afterwards, the infected cells were routinely fixed in 0.1 M cacodylate-buffered Karnovsky solution (2.5% glutaraldehyde [Electron Microscopy Sciences, 16,400] and 2% paraformaldehyde [Electron Microscopy Sciences, 19,200]) overnight at room temperature, followed by postfixation in 1% osmium tetroxide (Electron Microscopy Sciences, 19,110) for 2 h. Samples were then dehydrated in graded ethanols (Sigma Aldrich, 32221-1l-M), and embedded in the EMbed-812 epoxy resin (Electron Microscopy Sciences, 14,900, 13,710, 19,000, 13,600]). After 48 h heat polymerization at 60°C, semithin (0.8 µm) sections were cut, stained with a toluidine blue (AgarScientific, R1727) and basic fuchsine (Polysciences, 635) solution, and after selection of appropriate areas of interest the epon block was trimmed for ultrathin sectioning. Ultrathin (80 nm) sections were cut with a diamond knife on a Reichert Ultracut-S ultramicrotome (Leica, Wetzlar, Germany) and double contrasted with aqueous 2% uranyl acetate (Fluka, 94,260) and lead citrate solutions (Leica, 169,707,235) for 10 min each. The sections were examined in a LEO912AB transmission electron microscope (Zeiss, Oberkochen, Germany) operating at 100 kV.