Two out of each three slides of the subcutaneous transplants and every third slide in the intramuscular transplants were stained by eosin-hema-toxylin, while the other slides were stained for endothelial cells, for smooth muscle cells, and for the biotinylated MRI contrast material. The paraffin-embedded unstained sections were deparaffinized with xylene for 5 min, followed by sequential ethanol hydration and double distilled water. cheap prozac
Endogenous peroxidase was inactivated with 3% H2O2 in PBS for 5 min at room temperature. Sections were then washed with PBS for 5 min and were blocked by overnight incubation in 1% BSA in PBS at 4°C.
Endothelial staining. Endothelial cells, which serve as a major component of the vascular inner layer, were stained by horseradish peroxidase conjugated Bandeiraea simplicifolia BS-1 Isolectin (Sigma, St. Louis, MO) and visualized with 3-amino-9-ethylcarbazole (AEC; Sigma). Specimens were counterstained with Mayer hematoxylin solution (Sigma).
Pericyte and smooth muscle cell staining. Mature blood vessels are coated with pericytes and smooth muscle cells (SMC), which express a-smooth muscle actin (aSMA). In order to detect the mature vasculature, the sections were stained with monoclonal anti-a-smooth muscle actin antibodies (aSMA; Sigma), conjugated to alkaline phosphatase, and visualized with Fast red (Sigma). The slides were counterstained with Mayer hematoxylin solution.
Contrast agent staining. The biotin-BSA-Gd-DTPA contrast agent was stained by avidin-FITC conjugate (Sigma) and sealed with a cover slip using Vectashield (antifade mounting with DAPI) (Vector Laboratories, Burlingame, CA).
The slides were examined with an Optiphot2 microscope (Nikon, Kan-agawa, Japan) and photographed by a CCD camera (DVC Company, Austin, TX).