Perfusion Protocol

Animals are sacrificed via an intraperitoneal (IP) injection of 2 mL of 50 mg/mL sodium pentobarbital (Nembutal) and transcardially perfused with 500 mL of 10% buffered formalin. As soon as the animal stops breathing and is non-responsive to the paw pinch test the perfusion can begin. While waiting, close off the perfusion fluid reservoir and fill with formalin. Attach 18 G perfusion needle and open reservoir, flushing system. Clamp the tubing near perfusion needle.

A midline incision 1 cm inferior to the sternum and running cephalad is made with Metzenbaum (Metz) or Iris scissors. The diaphragm is dissected with the scissors, and two incisions are made from the midline incision laterally across the ribs to the axilla. The anterior chest wall is retracted using a small clamp at the distal sternal cartilage. The pericardium is bluntly dissected and the heart is freed. The 18 G perfusion needle is inserted in the left ventricle and clamped in place; the needle should go in up to just past the bevel. The clamp on the tubing is released. Quickly use the Metz to open the right atrium. For best results make sure that the exanguinating blood does not pool in the pleural cavity but is allowed to drain, either by cutting a canal in the lateral chest wall or by tilting the animal.

Tumors are dissected bluntly and with scissors. Upon removal they should be sketched in the lab notebook. The sketch should be annotated with orienting information based on scout images and observations during resection. Tumors are post-fixed in 10% buffered formalin for 24 hours, and then transferred to a 90% ethanol solution prior to sectioning. The sections of tumor were again transferred to 10% buffered formalin, dehydrated by alcohol, embedded in paraffin, and sectioned at a thickness of 3 ¿m. The tissue sections were stained with hematoxylin and eosin for routine pathology. Immunohistochemistry staining was conducted by using commercially available antibodies directed against vascular endothelial growth factor (VEGF). The bound primary antibodies were detected by the DAKO Labeled Streptavidin Biotin 2 system. Hematoxylin stain was used as a counter stain in the immunohistochemical staining process.