5 bromo 4 chloro 3 indolyl β d galactoside x gal

SA-β-gal activity was examined as described previously^{9 (link)}. Briefly, cells were fixed with 0.25% Glutaraldehyde in PBS at room temperature for 15 min, then washed with PBS 1 time, followed by incubation for 1–2 h at 37 °C in β-gal staining solution containing 1 mg ml^–1 5-bromo-4 chloro-3-indolyl-β-D-galactoside (X-gal, Takara), 5 mmol l^–1 potassium ferrocyanide, 5 mmol l^–1 potassium ferricyanide, 150 mmol l^–1 NaCl, 2 mmol l^–1 MgCl₂, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40. Then the stained cells were taken 5 photomicrographs with BZ-9000 or BZ-X810 (Keyence). Quantification of SA-β-gal activity was performed with the ImageJ program (version 1.53a). Atherosclerotic plaque was estimated by assessing sections with Oil Red O staining. Briefly, whole aortas were dissected to remove adventitial fat, opened, and pinned flat for fixing in 4% paraformaldehyde for 12 h at room temperature. Then the pinned aortas were washed for 1 min with 60% isopropyl alcohol and incubated in 0.5% Oil Red O solution (Sigma-Aldrich) in 60% isopropyl alcohol) for 15 min at 37 °C for staining. Subsequently, the samples were briefly immersed in 60% isopropyl alcohol solution and then washed with double distilled water. After the Oil Red O-stained specimens were photographed, quantification of the plaque area was done with ImageJ.

5‐bromo‐4‐chloro‐3‐indolyl‐β‐d‐galactoside (X‐gal) was from Takara Bio. The stock solution contained 20 mg ml⁻¹ X‐gal in N, N‐dimethylformamide. Phos‐tag acrylamide AAL‐107 was purchased from Wako Pure Chemical. The stock solution contained 5.0 mM Phos‐tag acrylamide AAL‐107 in 3% (v/v) methanol. Bis(2‐hydroxyethyl)iminotris(hydroxymethyl)methane (Bis‐Tris) was from Wako Pure Chemical. Other chemicals were purchased from Sigma‐Aldrich, Wako Pure Chemical, Nacalai Tesque, and BD.