Cy3 conjugated goat anti rat

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).

Larval tissues were collected and treated at 110–120 hours after egg deposition. Larvae were dissected in 1xPBS at room temperature and fixed for 20 minutes in 4% formaldehyde and the immunostaining procedure was performed as previously described^{35 (link)}. After several washes in 1xPBS+0,3% Triton X-100, wing imaginal discs were mounted on microscopy slides with Fluoromount G. Subsequently, samples were analysed with TCS SL Leica confocal system. Digital images were assembled using the Adobe Photoshop software. The following primary antibodies were used: monoclonal mouse anti-ci 1:50 (DSHB) and anti-phosphoJNK 1:400 (Cell Signaling Technology) were detected with Cy3-conjugated goat anti-mouse 1:500 (Jackson); polyclonal rabbit anti-Awd^{12 (link)} 1:1000 was detected using Cy3-conjugated goat anti-rabbit 1:1000 (Jackson) or DyLight 647-conjugated goat anti-rabbit 1:500 (Jackson); rabbit anti-MMP1 1:50 (DSHB) was detected using Cy3-conjugated goat anti-rabbit 1:1000 (Jackson); anti-cleaved-Caspase3 1:100 (Cell Signaling Technology) was detected using Cy3-conjugated goat anti-rabbit 1:2000 (Sigma); anti-ξPKC 1:200 (Santa Cruz Biotechnology) was detected with Cy5-conjugated goat anti-rabbit 1:1000 (Jackson). DE-Cad 1:25 (DSHB) was detected using Cy3-conjugated goat anti-rat 1:1000 (Jackson).

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).