Fluorescent microscope system

To assess the pathological changes, vulvar paraffin sections were stained with haematoxylin and eosin. Immunohistochemistry was used to detect the expression of caspase-1. Tissue sections were dewaxed, hydrated, antigen repaired, membrane permeated, and blocked. After incubating with primary antibodies against caspase-1 and HRP-conjugated secondary antibody (Servicebio, Wuhan, China), the tissue sections were visualized by DAB, and counterstaining was performed with haematoxylin. For immunofluorescence staining, the slides followed the same procedure as immunohistochemistry until antigen was repaired. After blocking with 10% normal goat serum for 1 h, the tissue sections were incubated with primary antibodies and Alexa Flour 488 or Cy3-conjugated secondary antibody. The nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI). The images were detected by Olympus fluorescent microscope system.

Aortic tissue was harvested from all animals. AAA tissue was fixed in Histochoice (VWR), and paraffin embedded. Paraffin blocks were sectioned at 5 μm, and deparaffinized. Processing for antigen retrieval was performed with Sodium Citrate solution, pH 6.0, for 10 min. Tissue sections were blocked with 10% serum, and sections were incubated with primary antibody anti-CD68, 1:100 [Bio-Rad, MCA341GA]. Sections were then incubated with anti-mouse secondary antibodies conjugated with HRP (Cell Signaling), DAB peroxidase substrate kit (Vector Laboratories), and counter stained with hematoxylin, imaged using an Olympus fluorescent microscope system. To evaluate AAA tissue morphology and pathology, tissue sections were also evaluated using Hematoxylin and Eosin (H&E) and Mason Trichrome (MT). AAA wall tissue-stained sections were then analyzed and quantified by Image J software via color deconvolution and shown as percentage of stained area for specific regions of interest (ROI).

Aortic tissue was harvested from all animals. AAA tissue was fixed in Histochoice (VWR), and paraffin embedded. Paraffin blocks were sectioned at 5 μm, and deparaffinized. Processing for antigen retrieval was performed with Sodium Citrate solution, pH 6.0, for 10 min. Tissue sections were blocked with 10% serum, and sections were incubated with primary antibody anti-CD68, 1:100 (Bio-Rad, MCA341GA), CCR2, 1:200 (Novus-Bio, NBP1-48338), CD86 (Thermo-Fisher, #942-MSM-P0) and CD206 (Thermo-Fisher, #187004-1-AP). For Immunofluorescence, sections were incubated with donkey anti mouse (Alexa Fluor 647), and donkey anti rabbit (Cy3) (Jackson ImmunoResearch Laboratories). All sections were imaged using a Leica THUNDER Imager 3D tissue microscope system. Sections were then incubated with anti-mouse secondary antibodies conjugated with HRP (Cell Signaling), DAB peroxidase substrate kit (Vector Laboratories), and counter stained with hematoxylin, imaged using an Olympus fluorescent microscope system. To evaluate AAA tissue morphology and pathology, tissue sections were also evaluated using Hematoxylin and Eosin (H&E), Mason Trichrome (MT) and Verhoeff-Van Gieson (VVG), imaged using NanoZoomer, Supported by the Alafi Neuroimaging Laboratory. All AAA wall staining intensity was analyzed in 3 random ROIs, quantified with Image J, and normalized to the total area of the aortic wall media.