Fluorescence dye

To analyze human AGXT2 transgene expression cell slides with primary aortic cells were fixed with 1:1 acetone-methanol solution for 10 min at 4 °C, washed 3 × 2 min with ice-cold PBS and blocked with Dako Protein Blocking solution for 20 min at room temperature. Afterwards, chamber slides were incubated with 1:100 rabbit polyclonal anti-FLAG antibodies (Sigma-Aldrich, St. Louis, MI, USA, Catalog #7425) for 2 h at 37 °C, washed 3 × 2 min with PBS and subsequently incubated with 1:250 anti-rabbit antibodies coupled with fluorescence dye (The Jackson Laboratory, Bar Harbor, ME, USA) at room temperature for 1 h. Finally, slides were washed 3 × 2 min with PBS, stained with 1:1000 DAPI and mounted with Moviol. To demonstrate the expression of human AGXT2-FLAG transgene in endothelial cells 1:100 rat anti-CD31 antibodies (Biolegend, San Diego, CA, USA, Catalog # 102401) (as marker of endothelial cells^{33 (link)}), and 1:250 anti-rat secondary antibodies (The Jackson Laboratory, Bar Harbor, ME, USA) were used. Double staining of CD31 and FLAG-tagged transgene was performed in the same way.

At the indicated period after the aneurysm induction, 5-µm-thick frozen sections were prepared. After blocking with 3% donkey serum (#AB_2337258, Jackson ImmunoResearch, Baltimore, MD, USA), slices were incubated with primary antibodies, followed by incubation with secondary antibodies conjugated with a fluorescence dye (Jackson ImmunoResearch). Finally, fluorescent images were acquired using a confocal fluorescence microscope system (FV1000 or FV3000, Olympus, Tokyo, Japan).
The following primary antibodies were used: mouse monoclonal anti-CD68 antibody (#ab31630, Abcam, Cambridge, UK), rabbit polyclonal anti-myeloperoxidase (MPO) antibody (#ab9535, Abcam), rabbit polyclonal anti-tumor necrosis factor (TNF)-alpha antibody (#ab6671, Abcam), mouse monoclonal anti-smooth muscle α-actin (SMA) antibody (#M0851, Dako, Agilent, Santa Clara, CA, USA).
The following secondary antibodies were used; Alexa Fluor 488-conjugated donkey anti-mouse IgG H&L antibody (#A21202, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 488-conjugated donkey anti-rabbit IgG H&L antibody (#A21206, Thermo Fisher Scientific), Alexa Fluor 594-conjugated donkey anti-mouse IgG H&L antibody (#A21203, Thermo Fisher Scientific).