Pertex medium

Immunohistochemistry for CD31 was performed as previously described^{34 (link)}. Briefly, 7–9 µm thickness paraffin sections were cut, deparafinized, rehydrated using ethanol gradients and placed in PBS. Antigen recovery was performed using a proteinase K (Roche, France) digestion procedure and then endogenous peroxidase was quenched using 3% (v/v) hydrogen peroxide for 5 min at room temperature (RT). Tissues were blocked with 2% horse serum, in PBS, for 30 min at RT. Primary antibody against CD31 (NB100–2284, Novus Biologicals, Bio-Techne, France) was used at 1:100 in 2% (w/v) BSA in PBS, and incubated overnight at 4 °C. After two washes with PBS, the anti rabbit Impress kit (VectorLabs, USA) was used according to the manufacturer’s instructions. Specific staining was obtained using the 3,3′-diaminobenzidine staining solution (VectorLabs, USA). Counterstaining was performed using Mayer’s haematoxylin. Samples were then mounted using Pertex medium (Sigma). Sample imaging was performed using a microscope (Nikon Eclipse 80i) equipped with a digital camera (Nikon Dxm 1200C). Sample analysis was performed at three different sample positions and a total of six animal samples were assessed per time point and condition. Vessel quantification was performed using ImageJ analysis, and the number of vessels was determined and normalized to the area of the defect.