Axioobserver d1 inverted fluorescent microscope

After anaesthesia (thiopental 120 mg/kg, i.p.), small fragments of lung and liver tissue were collected, washed in PBS solution, and then placed in 50 % OCT solution (SakuraTek, Japan) overnight for cryoprotection. Samples were then placed into OCT freezing compound and snap-frozen at -80^○C. OCT frozen liver and lung tissues were cut into 10 μm-thick cross-sections using a Leica CM1920 automatic cryostat and placed on poly-L-lysine-covered microscopic slides (Metzel Glaser Super Frost). Collected slides were immunostained using rabbit-anti-mouse NNMT (Abcam) primary antibody, followed by Cy3-conjugated goat-anti-rabbit secondary antibody (Jackson Immuno Research). Images were acquired using an AxioCam MRc5 digital camera and an AxioObserver.D1 inverted fluorescent microscope (Zeiss), stored as tiff files and analyzed automatically using Columbus software (version 2.4.2, Perkin Elmer).

Rings of the aorta were embedded in the OCT medium (Thermo) and frozen at −80°C using Leica CM1920 automatic cryostat (Leica, Wetzlar, Germany). The 5 μm thick cross-section slides were put on the microscopic glasses coated with poli-L-lysine and fixed for 10 min in 4% buffered formalin (Merck) and used for immunostaining of intercellular cell adhesion molecule 1 (ICAM-1). Aortic rings were preincubated with 5% normal goat serum (Jackson Immuno Research) and 2% dry milk in PBS, then immunostained using rat-anti-mouse ICAM-1 (eBioscience; 1:200) primary antibody overnight at 4°C. A secondary antibody Cy3-conjugated goat-anti-rat (Jackson Immuno Research; 1:300) was used, respectively, for 30 min at room temperature. Cell nuclei were visualized by Hoechst 33258 (Sigma; 1:1000) solution, and unspecific fluorescence of aortic elastic fibers were used as a background counterstaining channel. Images were acquired using an AxioCam HRm digital monochromatic camera and an AxioObserver.D1 inverted fluorescent microscope (Carl Zeiss). ICAM-1 fluorescence was calculated as the ICAM-1 expression area divided by the tissue area expressed as a percentage (ImageJ program).