Ki 67 nb500 170

The HaCaT cells were seeded in six-well confocal plates at a density of 1 × 10⁶/well. After 24 h of incubation, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 5 min at 37 °C. Then, the cells were incubated with 5% bovine serum albumin dissolved in PBS for 45 min at 37 °C. The HaCaT cells were incubated overnight with one of the following primary antibodies for immunocytochemistry: OR2AT4 (PA5-39811, 1:200; Thermo Fisher Scientific), sodium/potassium-ATPase α (Na⁺/K⁺-ATPase-α) (SC-48345, 1:50; Santa Cruz Biotechnology), and Ki-67 (NB500-170, 1:200; Novus). Then, the cells were washed three times with PBS. The HaCaT cells were incubated with anti-mouse IgG (Alexa Fluor 488; A11001, 1:500; Thermo Fisher Scientific) or anti-rabbit IgG (Alexa Fluor 532; A11009, 1:500; Thermo Fisher Scientific) secondary antibody for 1 h at room temperature. Next, the cells were incubated with 300 nM DAPI (Cayman Chemical, Ann Arbor, MI, USA) for 5 min in the dark. The cells were washed twice with PBS. Images were obtained using the LSM510 META confocal microscope and analyzed using LSM700 software (version 3.2; Carl Zeiss, Jena, Germany).

For cultured BMDM staining, the primary antibodies were as follows: Ki67 (NB500‐170, Novus Biologicals, Littleton, CO) and Galectin‐3 (Mac2) (ab76245, Abcam, Cambridge, MA). For staining of aortic root sections, the following primary antibodies were used: EGF‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) (PA5‐32399, Invitrogen), iNOS (ab3523, Abcam), CD206 (ab64693, Abcam), cleaved caspase‐3 (9661, Cell Signaling Technology (CST), Danvers, MA), CD68 (ab125212, Abcam), α‐actinin (ab137346, Abcam), CD3 (ab16669, Abcam), and Irf1 (8478, CST). The goat antirabbit secondary antibodies Alexa Fluor 647 (red; ab150115, Abcam) and Alexa Fluor 488 (green; ab150077, Abcam) were employed for immunofluorescent staining. A TUNEL Assay Kit (G3250, Promega, Madison, WI) was used to evaluate apoptosis levels in cultured BMDMs and aortic root sections. Cultured BMDMs and aortic root sections were also subjected to 4′,6‐diamidino‐2‐phenylindole (DAPI, Sigma) staining to identify nuclei. Images were captured using a RVL‐100 microscope.