Dm6b widefield fluorescent microscope

For immunohistochemical analysis, mice were anesthetized and aortas were carefully harvested after perfusion with PBS under a microscope. The heart with aorta was fixed with 4% paraformaldehyde (PFA)/5% sucrose in PBS solution 12 h at 4°C. The tissue samples were immersed in 15% sucrose solution for 12 h and then 30% sucrose solution for 24 h. The resulting specimens were embedded in Tissue-Tek OCT and frozen at −80°C and then sectioned with a cryostat as described previously (Yi et al., 2019 (link)). Briefly, serial sections (10 μm thick) of the aortic roots were collected (10–12 sections per mouse) starting at the appearance of aortic valves. The distance between each section was 150 μm, and totally of 100–120 serial cross-sections were obtained. To determine the immune cell populations in aortic lesions, the slides with multiple frozen aortic root sections were fixed in acetone and washed twice with PBS. Antibodies were performed on consecutive cross-sections for Treg cells (anti-Foxp3, 1:500, Abcam). Slides were stained using the Tyramide Signal Amplification kits in MHPL core facility of Northwestern University. All slides containing the cross-sections were digitally imaged with Leica DM6B widefield fluorescent microscope. An in-house software written in Python was used for automated and quantitative image analysis (Yi et al., 2019 (link)).