Ls f20750

RIPA buffer was used to lyse cells to collect proteins. ELISA for human HDAC3 (LS-F55924, LSBio, Seattle, WA, USA), human enhancer of zeste homolog 2 (EZH2; LS-F7278, LSBio), human DNA Methyltransferase 1 (DNMT1; LS-F7340, LSBio), human sirtuin 1 (SIRT1; LS-F12606, LSBio), human jumonji domain containing 3 (JMJD3; LS-F74347, LSBio), mouse IL-6 (Ab100713, Abcam), mouse IL-1β (Ab197742, Abcam), mouse tumor necrosis factor alpha (TNFα; Ab208348, Abcam), mouse interferon gamma (IFNγ; Ab282874, Abcam), mouse arginase 1 (ARG1; Ab269541, Abcam), mouse CD163 (Ab272204, Abcam), mouse α-smooth muscle actin (α-SMA; NBP2-66429, Novus Biologicals, Shanghai, China), Vimentin (LS-F7624, LSBio, Seattle, WA, USA) and mouse Collagen IV (LS-F20750, LSBio) were utilized as per the manufacturer’s instructions.

Brain tissues were homogenized in RIPA buffer (MilliporeSigma) containing complete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). After centrifugation (100,000g for 1 hour at 4°C), the supernatant was used for biochemical analysis as described (53 (link)). ApoE (53 (link), 55 (link)), collagen IV (LS-F20750, LSBio), MMP-2 (MMP200, R&D Systems), and MMP-9 (NBP2-60095, Novus Biologicals) in brain lysate were measured by ELISA. Brain lysate measurements were normalized to total protein concentrations determined by BCA assay (Thermo Fisher Scientific). Total cholesterol and triglycerides in plasma were measured using a Cholesterol Assay Kit (A12216, Thermo Fisher Scientific) and Triglyceride Assay Kit (ab65336, Abcam), respectively. Some brain samples were subjected to Western blotting using anti–Iba-1 antibody (17198, Cell Signaling Technology) and anti–β-actin antibody (3700, Cell Signaling Technology), followed by quantification through LI-COR Odyssey.