Immunohistochemistry
Four weeks after the transplantation, cardiac grafts were cut horizontally into 4 blocks, embedded in OTC compound (Sakura Finetechnical Co, Tokyo, Japan) and kept at −80°C until staining. Nine slices (3 slices from 3 blocks from the apex) were made for immunostaining with a kit (Histofine SAB-PO kit, Nichirei Co, Tokyo, Japan) for macrophages (MOMA-2) and CD4- and CD8-positive T cells, as previously described.19 Three fields where a coronary artery was recognized in the center were selected from each animal to count the number of macrophages and calculate a percent-positive area in a blind manner at a magnification of ×200.
