Anti mouse secondary antibody conjugated to alexa fluor 594

Immunofluorescence staining was performed as previously described [14 (link)]. Briefly, frozen section slides were thawed, fixed with 10 % paraformaldehyde, blocked in 3 % bovine serum albumin (BSA), and incubated with primary antibody to α-smooth muscle actin (α-SMA) at a 1:500 dilution (Abcam, Cambridge, UK) and isolectin B4 conjugated to Alexa Fluor 647 at a 1:100 dilution (Thermo Fisher Scientific, Waltham, MA) overnight at 4 degrees Celsius. After rinsing with PBS, anti-mouse secondary antibody conjugated to Alexa Fluor 594 at a 1:200 dilution (Cell Signaling, Danvers, MA) was applied and allowed to incubate for 1 h at room temperature. Slides were rinsed, and DAPI was applied for 5 min, followed by rinsing and mounting. Images were analyzed at 20× magnification with an Olympus VS200 Slide Scanner (Olympus Corporation, Tokyo, Japan). Image analysis was performed with QuPath software in a blinded fashion [15 (link)]. Capillary density was determined by defining positive isolectin B4 staining by thresholding and determining percent of tissue area stained. Arteriolar count was determined by defining positive α-SMA staining by thresholding and determining the number of objects with a minimum size of 100um² per area of tissue section. Catalog numbers are listed in Supplemental Table 1.