Aortic Immunofluorescent Staining Protocol

Abdominal aortas remained intact for en face staining with ICAM-1 (Abcam) and VCAM-1 (Abcam) or aortas were dissected until 3–5 mm before the iliac bifurcation to obtain transverse sections. For the latter, aortas were embedded in paraffin for subsequent cutting sections into 10 μm slices for immunofluorescent staining. Next, the transverse sections were stained as described previously (Schnitzler et al., 2020 (link)). In summary, the following antibodies were used: ICAM-1 (Abcam), GLUT-1 (Thermo Fisher) and secondary antibody Alexa Fluor 568 for PFKFB3 and Alexa 633 for GLUT1-1 (both Thermo Fisher). HIF1α (R&D systems, MAB1536), GLUT3 (Invitrogen, MA5-32697), and secondary Alexa Fluor 647 for HIF1a and Alexa Fluor 488 for GLUT3 (both Thermo Fisher Scientific). The transverse sections aortas were fixed with DAKO mounting medium containing DAPI (Agilent, Santa Clara, United States). All samples were visualized on a Leica TCS SP8 Confocal laser scanning microscope and quantified using ImageJ.