Modified histological staining for the identification of arterial and venous segments of brain microvessels

This study aimed to develop a modified histochemical staining technique to successfully identify arterial and venous segments of brain microvessels. Gelatin/red ink-alkaline phosphatase-oil red O (GIAO) staining was developed from the traditional gelatin–ink perfusion method. Oil red Chinese ink for brush writing and painting mixed with gelatin was used to label cerebral vascular lumens. Subsequently, alkaline phosphatase staining was used to label endothelial cells on the arterial segments of cerebral microvessels. Thereafter, the red ink color in vessel lumens was highlighted with oil red O staining. The arterial segments of the brain microvessels exhibited red lumens surrounded by dark blue walls, while the venous segments were bright red following GIAO staining. Meanwhile, the nerve fiber bundles were stained brownish-yellow, and the nuclei appeared light green under light microscope. After cerebral infarction, we used GIAO staining to determine angiogenesis features and detected notable vein proliferation inside the infarct core. Moreover, GIAO staining in conjunction with hematoxylin staining was performed to assess the infiltration of foamy macrophages. Red Chinese ink enabled subsequent multiple color staining on brain section. Oil red O was introduced to improved the resolution and contrast between arterial and venous segments of microvessels. With excellent resolution, GIAO staining effectively distinguished arterial and venous segments of microvessels in both normal and ischemic brain tissue. GIAO staining, as described in the present study, will be useful for histological investigations of microvascular bed alterations in a variety of brain disorders.