Ctr 6500

For immunofluorescence, cultured MSCs were fixed in 4% paraformaldehyde (PFA) and processed as described previously^{58 (link)}. Cells were stained with primary and secondary antibodies listed in Table S1. For immunohistolocalization assays, freshly collected fetal SGs and adult SMGs were fixed and embedded as described previously^{59 (link)}. 10 µm cryostat sections were subsequently stained with primary and secondary antibodies (Table S1) and were counterstained with ProLong® Gold antifade reagent with 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Grand Island, NY). Images were captured on Leica CTR6500 (Leica Microsystems, Buffalo Grove, IL) and EVOS epifluorescence microscopes (Thermo Fisher Scientific, Waltham, MA). Images were further analyzed using NIH-ImageJ software (NIH, Bethedsa, Maryland). Quantification of the images was done using NIH-ImageJ software to analyze 10 to 15 random images at 20x magnification from ≥3 fetal and adult SMG sections.

Pieces of mouse liver were fixed, embedded, sectioned with a cryostat and stained as previously described [30] (link). Primary and secondary antibodies used to stain the sections are listed in Table S1. Slides were covered with ProLong Gold antifade reagent with 40′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Grand Island, NY, USA). Images were analyzed with a Leica CTR6500 (Leica Microsystems, Buffalo Grove, IL, USA). Colocalization analysis was performed using iVision software (BioVision Technologies, Exton, PA, USA).

Cell morphology following stress treatment was visualized by fixing the cells immediately after stress and staining for F-actin, a cellular skeleton protein, using rhodamine phalloidin (Invitrogen). Cells were fixed with 3.7% paraformaldehyde in a phosphate buffered solution (PBS) (Fisher Scientific) and permeabilized using 0.1% Triton X-100 (Sigma)/PBS. For blocking, samples were incubated in 1% bovine serum albumin (Amersham) dissolved in PBS for 30 minutes at room temperature followed by 20-minute incubation in rhodamine phalloidin solution in the dark. For nucleus counterstaining, cells were mounted with VECTASHIELD mounting medium with DAPI (DAPI: 4′,6-diamidino-2-phenylindole) (Vector Laboratories). Stained images were acquired using a fluorescent inverted microscope (CTR6500, Leica Microsystems).

Immunohistochemical analyses were performed as previously described [19 (link)]. Briefly, at the indicated period after aneurysm induction, 5-um-thick frozen sections were made as described above. After blocking with 3% donkey or goat serum (Jackson ImmunoResearch, Baltimore, MD, USA), slices were incubated with primary antibodies followed by incubation with fluorescence-labeled secondary antibodies (Jackson ImmunoResearch). Finally, fluorescent images were acquired through a confocal fluorescence microscope system (CTR6500, Leica Microsystems, Tokyo, Japan). Representative images from at least 3 independent samples are shown in Figures 1 and 2. The relative intensity of positive staining in IA walls from each experiment was measured by imaging software and statistically analyzed.
The following primary antibodies were used: mouse monoclonal anti-smooth muscle alpha actin antibody (Thermo scientific, Waltham, MA, USA), rabbit monoclonal anti-phospho NF-kappaB p65 (ser536) antibody (Cell Signaling Technology, Danvers, MA, USA), rat monoclonal anti-TNF-alpha antibody (Lifespan Bioscience, Seattle, WA, USA), goat polyclonal anti-MCP-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-COX-2 antibody (Cayman Chemicals, Ann Arbor, MI, USA), rat monoclonal anti-F4/80 antibody (Abcam, Cambridge, UK).

Comet assay was conducted using a Comet Assay Kit (Abcam, catalog no. ab238544). Briefly, cells treated as indicated were harvested and resuspended at a density of 1 × 10⁵ cells/ml in PBS. Under low lighting, samples were combined with comet agarose and spread onto a precoated base layer. Slides were immersed into lysis buffer in the dark at 4 °C for 1 h and transferred to an electrophoresis chamber for 30 min. The slides were then immersed in distilled H₂O, followed by 70% ethanol. Upon air drying, Vista Green DNA Dye was added for 15 min. Images were acquired by epifluorescence microscopy (CTR6500; Leica Microsystems Inc.), and DNA damage was analyzed by CometScore 2.1 (TriTek Corp).