Axioobserver d2 inverted fluorescent microscope

Lungs were fixed in 4% buffered formalin (for at least 48 h). After macroscopic analysis of the number of metastases, the lungs were prepared using the paraffin method, cut into 6 μm sections on an Accu-Cut® SRM™ 200 Rotary Microtome and stained with hematoxylin and eosin.
Light microscopic examination and photographic documentation were performed using an Olympus BX53F microscope equipped with a digital camera. Pictures were taken under the magnification 20× and 200×.
For immunohistochemical staining of VCAM-1 and vWF in the lung vasculature following deparaffinization, sections were pretreated according to the citrate-based HIER protocol and then preincubated with 5% goat serum (Jackson ImmunoResearch) and 2% dry milk to minimalize non- specific binding of antibodies. Primary rat-anti-mouse VCAM-1 (Chemicon) or rabbit-anti-mouse vWF (Abcam) antibodies were used, followed by Cy3-conjugated goat-anti-rat or Cy3- conjugated goat-anti-rabbit secondary antibodies (Jackson ImmunoResearch), respectively. Images were acquired using the AxioObserver D2 inverted fluorescent microscope (Carl Zeiss) and an AxioCam HRm monochromatic digital camera and stored as TIFF files. Fluorescence intensity was analysed automatically by Columbus software (Perkin Elmer).

Aortic rings were placed perpendicularly in OCT compound (Thermo) and then snap-frozen at − 80 °C. The blocks were mounted on the cryostat holder and cut into 10-μm-thick cross-sectional slides using the Leica CM1950 automatic cryostat. The sections were placed on polylisine-covered (Sigma-Aldrich) microscopic slides (Super Frost, Mentzel Gläser), and then acetone fixed (10 min). Pre-incubation with 2.5% horse serum (Vector Labs) and 2% dry milk was performed to minimize non-specific binding of antibodies. For indirect immunohistochemical detection of von Willebrand factor in endothelium, sections were incubated inside humid chambers with polyclonal rabbit anti-mouse vWF Ig (Abcam) following rinsing in PBS secondary biotinylated horse anti-rabbit Ig (Vector Labs). After another rinse in PBS, sections were incubated with Cy3-conjugated streptavidin (Jackson ImmunoResearch), and then mounted in glycerol-PBS. Images of the immunostained sections were acquired using the AxioObserver D2 inverted fluorescent microscope (Carl Zeiss) connected to a AxioCam HRm monochromatic digital camera, and stored as TIFF files. Fluorescence parameters were analysed automatically by Columbus software (Perkin Elmer).