Immunohistochemical Analysis of Aortic Inflammation

Mouse aortic sinus was serially cut in 7 μm transversal sections, as previously described^{11 (link),68 (link)}. Sections from mouse specimens were fixed in acetone and immunostained with specific antibodies anti-mouse CD68 (macrophages, ABD Serotec, Düsseldorf, Germany), anti-mouse Ly-6G (neutrophils, BD PharmingenTM, San Jose, CA, USA), anti-mouse MMP-9 (R&D Systems). Vector Red alkaline phosphatase substrate: SK-5100; in association with Levamisole solution: SP-5000; which produce a magenta coloration is used for revelations (Vector Laboratories, INC, CA, USA). Quantifications were performed using the MetaMorph or Definiens software. Results for other parameters were calculated as percentages of stained area on total lesion area, number of infiltrating cells per mm2 of lesion area or number of lymphatic vessels in adventitia.

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Santiago-Raber M.L., Montecucco F., Vuilleumier N., Miteva K., Baptista D., Carbone F., Pagano S., Roth A., Burger F., Mach F, & Brandt K.J. (2020). Atherosclerotic plaque vulnerability is increased in mouse model of lupus. Scientific Reports, 10, 18324.

Publication 2020

anti-mouse CD68 anti-mouse Ly-6G anti-mouse MMP-9 Vector Red alkaline phosphatase substrate: Levamisole solution

Acetone Adventitia Alkaline phosphatase Anti antibodies Aortic sinus Levamisole Lymphatic vessels Macrophages Magenta Mmp 9 Mouse Neutrophils Vector

Corresponding Organization :

Other organizations : University of Geneva, Ospedale Policlinico San Martino, University Hospital of Geneva, Foundation for laboratory medicine

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Variable analysis

independent variables

Serial cutting of mouse aortic sinus in 7 μm transversal sections

dependent variables

Quantification of CD68+ (macrophages), Ly-6G+ (neutrophils), and MMP-9+ cells
Percentage of stained area on total lesion area
Number of infiltrating cells per mm2 of lesion area
Number of lymphatic vessels in adventitia

control variables

Mouse specimens
Fixation of sections in acetone
Immunostaining with specific antibodies
Use of Vector Red alkaline phosphatase substrate and Levamisole solution for revelation

controls

Positive controls not explicitly mentioned
Negative controls not explicitly mentioned

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