Fig. 5. (A) NADPH-diaphorase in the icefish ventricle. (Bottom) Representative image showing the localization of NOS in paraformaldehyde-fixed transverse ventricular sections (5 µm) processed as described in Materials and methods. Note the dark blue reaction product detecting NOS activity in endocardial endothelial (EE) cells (arrowhead) and cardiomyocytes (arrow) (100x). (Top) Control image obtained by incubating transverse ventricular sections in absence of NADPH (100x). (B) iNOS immunofluorescence. Frozen transverse ventricular sections (7 µm) were incubated with 1:100 anti-iNOS antibody as described in Materials and methods. Fluorescent immunolabeling of iNOS in the icefish ventricle is densely localized in the cytoplasm of the myocardiocytes (white arrowhead). Note the absence of iNOS in the subepicardial layer (white arrow) and in the EE cells (yellow arrow). Bar, 40 µm.