Af594 gt α rb

Wounds from the excisional model were harvested upon closure (day 24) and immediately fixed in 4% paraformaldehyde overnight, dehydrated with sequential ethanol concentrations (30, 50, 70, and 95%), xylene, and paraffin washes, and embedded in paraffin for sectioning. To evaluate dermal integrity, wound sections were stained with Masson’s Trichrome and visualized using light microscopy. To assess vasculature in the healed wounds, sections were stained for CD31 (1° - 1:200 Rb α CD31, Ab28364, Abcam; 2° - 1:200 AF488 Gt α Rb, Life Technologies, Carlsbad, CA, USA) and VEGF-A (1° - 1:200 Rb α VEGFA, Ab46154, Abcam; 2° - 1:200 AF594 Gt α Rb, Life Technologies). To assess superoxide protein levels in the healed wounds, sections were stained for SOD2 (1° - 1:200 Rb α SOD2, Ab13533, Abcam; 2° - 1:200 AF594 Gt α Rb, Life Technologies). To assess the presence of transplanted cells in the healed wounds, sections were stained for human nuclei (α -Nuclei, AF488 conjugate, MAB1281A4, EMD Millipore, Burlington, MA). For IHC analysis, nuclei were stained with DAPI, and Adobe Photoshop CS6 (Adobe Systems) was used to superimpose the images taken with the same settings. Stained pixels per high magnification field were quantified using ImageJ.