Quantifying Microvascular Density in Tissue

Microvessel density was determined by immunofluorescent staining, which was performed as previously described [22 (link)]. Briefly, frozen sections were thawed, fixed with 10% paraformaldehyde, blocked in 3% bovine serum albumin, and incubated overnight at 4 °C with primary antibody to α-smooth muscle actin (Abcam, Cambridge, UK) for arteriolar staining and isolectin B4 conjugated to Alexa Fluor 647 for capillary staining (Thermo Fisher Scientific, Waltham, MA, USA). Slides were rinsed with PBS and incubated with an anti-mouse secondary antibody conjugated to Alexa Fluor 594 (Cell Signaling, Danvers, MA, USA) at room temperature for one hour. Slides were rinsed, and DAPI was applied for five minutes, then rinsed again and mounted. Images were analyzed at 20× magnification with an Olympus VS200 Slide Scanner (Olympus Corporation, Tokyo, Japan). Image analysis was performed with QuPath software [23 (link)]. Capillary density was determined by thresholding positive isolectin B4 staining and determining the percent of tissue area stained. The arteriolar count was determined by thresholding positive SMA staining and determining the number of objects with a minimum size of 100 µm² per area of tissue sections.