Ab269513

As previously described [13 (link)], the expression of NF-қB p65 (6956T, CST, Boston, MA, USA), α-SMA (ab5694, Abcam, Cambridge, MA, USA), CD68 (ab201340, Abcam, Cambridge, MA, USA), TFRC (ab269513, Abcam, Cambridge, MA, USA), and FTL (ab269513, Abcam, Cambridge, MA, USA) in the abdominal aorta tissues were assessed via immunostaining. Briefly, sections were blocked with a 3% hydrogen peroxide (H₂O₂) to block the endogenous peroxidase. Then, the sections were incubated overnight with primary anti-NF-қB p65, anti-α-SMA, anti-TFRC, and anti-FTL antibodies at 4 °C. Next, washing was performed and the secondary antibody was incubated for 1 h at room temperature. After washing, sections were counterstained with hematoxylin and visualized with a diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA, USA). For immunofluorescence staining, the sections were incubated with primary anti-CD68 overnight at 4 °C. Afterwards, the sections were incubated with the appropriate fluorescently labeled secondary antibodies for 1 h at room temperature. After washing, DAPI (Roche) was used to stain the nuclei for 8 min. Images were taken with a DS-Fil digital camera mounted on a Nikon Eclipse 80i fluorescence microscope (Nikon).

The injured CAs underwent a series of processes, including excision, fixation, and embedding in paraffin. Then, the CAs were cut into 4 μm thick sections and analyzed through IF staining. Homoplastically, the treated HUVECs were fixed in 4% paraformaldehyde. Next, as previously described [20 (link)], the sections and HUVECs were incubated with primary antibodies against cathepsin B (1 : 100, ab214428, Abcam), cathepsin D (1 : 200, #2284 s, Cell Signaling Technology), ferritin (1 : 50, ab75973, Abcam), and TfR (1 : 50, ab269513, Abcam) at 4°C overnight. After washing three times on the following day, the samples were then incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32790, Invitrogen, Carlsbad, CA, USA), Alexa Fluor 555-conjugated donkey anti-rabbit IgG (H+L) (1 : 300, A32794, Invitrogen), Alexa Fluor 488-conjugated goat anti-mouse IgG (1 : 300, A-11001, Invitrogen), and Alexa Fluor 555-conjugated goat anti-mouse (1 : 300, A-21424, Invitrogen) secondary antibodies at 37°C for 1 h. Next, 4,6-diamino-2-phenylindole (SouthernBiotech, Birmingham, AL, USA) was added to each section for coverslipping. Finally, the sections were observed under a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Tokyo, Japan), and ImageJ software was used to quantify the fluorescence intensity.