Immunohistologic labeling of murine endothelium

Reliable identification of endothelial cells is a prerequisite for understanding vascularity changes in many cardiovascular diseases and therapeutic interventions. With the rising use of mouse models of disease and genetic manipulation, a consistent system to label murine endothelial cells in normal and diseased tissues would be an invaluable tool.

Immunohistologic and histochemical methods were used to label endothelial cells in normal and infarcted heart as well as small intestine. Isolectin B(4) or antibodies to thrombomodulin, vWF, Tie-2, VE-cadherin, CD34, and CD31 were used to immunostain tissues fixed in either 4% formaldehyde (prepared fresh from powdered paraformaldehyde, PF), methyl Carnoy's (MC), zinc (Zn) (all paraffin-embedded), or frozen sections. In addition, we used beta-galactosidase histochemistry in frozen sections from the Tie-2/beta-galactosidase transgenic mouse, in which the lacZ reporter gene is driven by the endothelial-specific Tie-2 promoter.

Immunohistologic localization of endothelial cells was best accomplished using anti-CD31 in Zn-fixed, paraffin-embedded tissues. Antithrombomodulin staining also worked in Zn-fixed tissues as well as frozen tissues, but additional steps were required to reduce background. The beta-galactosidase reporter system also marked endothelial cells in frozen Tie-2 transgenic mouse tissues; however, this system required careful control of fixation and optimization of histochemistry and was not amenable to double labeling. Lectin staining gave complete labeling of endothelial cells but cross-reacted intensely with macrophages in the infarcted heart. We conclude that anti-CD31 immunostaining in Zn-fixed, paraffin-embedded murine tissue offered superior morphology and permitted optimal identification of proliferating endothelial cells during infarct repair.