0.1 m cacodylate buffer

Neutrophils were harvested, seeded on glass cover slips, and stimulated with either RPMI, MβCD, or Simvastatin as stated above. After 3 h incubation under either hypoxia or normoxia, cells were fixed in 1.5% glutaraldehyde (Sigma-Aldrich Chemie GmbH, St. Louis, MO, USA) and 3% PFA, buffered with 0.1 M cacodylate buffer (Serva Electrophoresis, Heidelberg, Germany) for 24 h and subsequently washed with 0.1 M cacodylate buffer. For further processing, the samples were embedded with 1% osmium tetroxide (Science Services GmbH, Munich, Germany) and dehydrated in a series of graded ethanol, followed by critical-point-drying and coating with gold in a sputter-coater (SCD040, Oerlikon Balzers), as described previously [44 (link),45 (link)] Afterward, the samples were mounted on 0.5” Aluminum Specimen Stubs (Agar Scientific, Stansted, Essex, UK) using 12 mm Leit-Tabs (Plano) and examined using a Zeiss EVO 15 scanning electron microscope (Carl Zeiss Microscopy, Oberkochen, Germany) operating with an acceleration voltage of 10 kV.

The tissue from the ex vivo and in vivo experiments was fixed in a mixture of 2% paraformaldehyde (Merck KgaA, Germany) and 2% glutaraldehyde (Serva, Germany) in 0.1 M cacodylate buffer (pH 7.4; Serva, Germany) for 3 to 4 h at 4 °C. The tissue samples were then rinsed in 0.1 M cacodylate buffer and post-fixed in 1% osmium tetroxide (Serva, Germany) in the same buffer for 1 h at 4 °C. Specimens were critical-point dried, attached to aluminum holders with silver paste (Silver DAG1415, Plano GmbH, Germany), sputter-coated with gold, and examined in a Tescan Vega3 scanning electron microscope at 30 kV (Brno, Czech Republic).

To assess ultrastructural morphology, glutaraldehyde-fixed PCLSs (8 slices derived from 3 dogs) were rinsed in 0.1 M cacodylate buffer (Serva Electrophoresis GmbH, Heidelberg, Germany) for 5 h on a roll mixer, followed by postfixation with 1% osmium tetroxide (Carl Roth GmbH + Co. KG, Karlsruhe, Germany). After dehydration through a series of graded alcohols, PCLSs were embedded in epoxy resin as described previously [42 (link)]. Selected representative sections were prepared as ultrathin sections, were contrasted with uranyl acetate and lead citrate, followed by evaluation by transmission electron microscopy (Carl Zeiss LEO EM 906, Leo Elektronenmikroskopie GmbH, Oberkochen, Germany).