Nis elements ar imaging software 4

Immunofluorescent staining was performed as previously described [65 (link)]. The primary antibodies used were as follows: cGAS (Mouse, 1:100, sc-515777, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MOMA-2 (Rat, 1:100, ab33451, Abcam, Cambridge, UK), CD31 (1:100, AF3628, R&D Systems, Minneapolis, MN, USA) and α-SMA (Rabbit, 1:100, GB13044, Servicebio, Wuhan, China). 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) was applied to stain nuclei. Fluorescent images were captured with a Nikon A1Si confocal microscope (Nikon, Tokyo, Japan), and analyzed with NIS Elements AR Imaging Software 4.10 (Nikon) and ImageJ 1.41 software (NIH, Bethesda, MD, USA).

Serial sections (6 μm thick) of the aortic root (3–5 sections per mouse) were stained with hematoxylin and eosin (HE), Oil Red O (ORO), or Masson’s trichrome (MASSON) and the microscopic images were then collected. Quantitative immunostaining was performed using primary antibodies for capase-3 (1:200, Cell Signaling Technology [CST], Danvers, MA, USA), Mac-3 (1:200, Abcam, USA), MOMA-2 (1:200, Abcam, USA), or α-smooth muscle actin (α-SMA, 1:100, Abcam, USA). Fluorescently labeled secondary antibodies were used for detection. The quantification of colocalized signals and the percentage of positive area in the images were performed using NIS Elements AR Imaging Software 4.10 (Nikon) or ImageJ 1.41 software (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA).

Immunofluorescent staining was performed as described^{47 (link),54 (link)}. All rats were transcardially perfused with PBS, then brain tissues were fixed with 4% PFA, and frozen blocks were cut into 8–20 μm sections. The following primary antibodies were used: p-MLKL (Rabbit, 1:50, ab187091; Abcam, UK), Iba1 (Mouse, 1:50, ab15690 and Goat, 1:50, ab5076; Abcam, UK), CD31 (Mouse, 1:50, ab119339; Abcam, UK), and TNF-α (Rabbit, 1:100, ab6671; Abcam, UK). The secondary antibodies are shown in Supplementary Table 1. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Samples were visualized with a Nikon A1Si confocal microscope (Nikon, Japan). Co-localization and percentage-positive area images were analyzed and performed using NIS Elements AR Imaging Software 4.10 (Nikon) and ImageJ 1.41 software.