Quantifying Ischemic Tissue Microvascular Density

Two weeks after surgery the mice were sacrificed using an overdose of pentobarbital 150 mg/kg/ml (via i.p. administration), and then systemically perfused animals were fixed with 4% paraformaldehyde as described previously [14 (link), 16 (link)]. Ischemic tissues were left in 4% paraformaldehyde overnight at 4°C, and anterior tibial muscles were excised, and embedded into paraffin. For evaluation of infarcted tissue microvascular density (MVD), heat-induced epitope retrieval was performed in deparaffinized tissue sections. Then, sections were incubated overnight with the antibody against anti-rabbit polyclonal CD34 antibody (1:200, clone: EP373Y, Abcam cat. # ab81289). To evaluate pericyte recruitment, Cy3-conjugated monoclonal anti-actin alpha-smooth muscle (α-SMA) antibody (1:200, clone: 1A4, Sigma-Aldrich) was used. The tissue sections for MVD and pericyte recruitment were observed and counted using fluorescence microscopy VH Analyzer (Keyence).