Quantifying Collateral Vessel Diameter and Macrophages

The collateral vessel was carefully removed to retain structural integrity in order to evaluate vessel diameter. Adipose tissue surrounding the collateral vessel was also harvested during the removal process and was used for macrophage quantification. Harvested collateral vessels and surrounding adipose tissue samples were stained concurrently with α-smooth muscle actin Cy3 (1:200, Clone 1A4, Sigma, St. Louis, MO) and Alexa Fluor 647 anti-mouse CD68 (1:200, Clone FA-11, AbD Serotec, Raleigh, NC) as described previously^{43 (link)}. Four unique FOVs were acquired for each sample as 40 µm z-stacks and the number of CD68⁺ cells were quantified using ImageJ. Data presented include vessel diameter measurements of the feeding collateral vessel, the first branch of the collateral vessel, and the second branch of the collateral vessel.