Epoxy resin

Sampling and processing of the lungs were carried out as described elsewhere [25 (link),38 (link),39 (link)]. Briefly, each lung was embedded in 2% aqueous agar—agar (Merck, Darmstadt, Germany) and cut from apex to base into parallel slices with a thickness of 2 mm using a tissue slicer. Slices from both lungs were alternatively taken for light and electron microscopy. Lung slices collected for electron microscopy were cut into tissue blocks with a size of 1–2 mm³. After additional immersion fixation, specimens for light and electron microscopy were rinsed repeatedly in cacodylate buffer. Postfixation followed in 1% OSO₄ in 0.1 M cacodylate buffer for 2 h. Again specimens were rinsed in cacodylate buffer then in distilled water and stained en bloc overnight at 4–8°C in half-saturated aqueous uranyl acetate solution. Specimens were dehydrated in ascending series of acetone. For ultrastructural analyses tissue blocks were embedded in epoxy resin (Serva Electrophoresis GmbH, Heidelberg, Germany). Prepared 70 nm thin ultra-thin sections were stained with lead citrate and uranyl acetate. For light microscopy whole tissue slices were embedded in the methacrylate resin Technovit 7100 (Heraeus Kulzer GmbH, Hanau, Germany). 1.5 μm sections were cut and stained with toluidine blue.

For electron microscopic analyses, different cell lines were fixed in freshly prepared 2% glutaraldehyde ELMI grade in phosphate buffered saline (PBS; both Sigma-Aldrich) for 2 hours at 4 °C, washed and stored in PBS. Subsequently, samples were post-fixed in 1% veronal-acetate-buffered OsO₄ (Merck) for 1 hour, dehydrated in a graded series of ethanol and embedded in epoxy resin (Serva). Ultrathin sections were stained in 1% uranyl acetate or alternatively, in 0.2% OTE (Oolong Tea Extract, Nisshin EM Co.), and subsequently in 8% alkaline lead citrate (both Merck), followed by examination in a transmission electron microscope at 80 kV (Tecnai 20, FEI Company).