*2.3. Myocardial Histology and Enzyme Histochemistry*

Conventional hematoxylin–eosin and Van Gieson staining of heart left ventricular tissue sections were used for a light microscopic examination of the myocardial structure (Zeiss Apotome 2 microscope: Carl Zeiss, Jena, Germany). Catalytic enzyme histochemistry was performed according to [21] using 10-μm-thick heart cryostat sections to demonstrate the activities of capillary endothelium-related alkaline phosphatase (AP, E.C.3.1.3.1) with naphthol AS-MX phosphate as a substrate and dipeptidyl peptidase-4 (DPP4, E.C.3.4.15.4) with glycyl-L-proline-4-methoxy-beta naphthylamide as a substrate to detect functional arteriolar and venular capillary network. For quantification of intensity of AP and DPP4 histochemical reactions (corresponding to the enzyme activity), randomly selected areas (15 per heart) of positive signal were analyzed and defined as the number of pixels with a code lower than 128 on the "0–255 RGB color scale". The total number of positive pixels was expressed as a total integral optical density per area (IOD) (Image-Pro Plus).
