Anti cd68

The following primary antibodies were used in this study: anti‐β‐actin (cat. no. 13E5; Cell Signaling Technology, Danvers, MA, USA), anti‐DJ‐1 (cat. no. ab76088; Abcam, Cambridge, UK), anti‐αSMA (cat. no. ab14106; Abcam), anti‐calponin (cat. no. ab46794; Abcam), anti‐osteopontin (cat. no. ab8448; Abcam), anti‐tropoelastin (cat. no. ab21600; Abcam), anti‐vimentin (cat. no. D21H3; Cell Signaling Technology), anti‐CD68 (cat. no. ab201340; Abcam), Anti‐ p44 / 42 MAPK, phospho (Erk1 / 2) Duet – PhosphoPlus (cat. no. 8201; Cell Signaling Technology) and anti‐Kruppel‐like factor 4 (KLF4; cat. no. ab214666 from Abcam; cat. no. D1F2 from Cell Signaling Technology). The following secondary antibodies were used in this study: rabbit/mouse Alexa Fluor 488 (cat. nos. A‐11034 and A‐11001; Thermo Fisher Scientific, Waltham, MA, USA) and rabbit/goat Alexa Fluor 594 (cat. nos. A‐11005 and A27016; Thermo Fisher Scientific). ERK inhibitor (SCH772984; Catalog No.S7101) was purchased from selleckchem (USA). Oil red O (cat. no. O0625) was purchased from Sigma‐Aldrich (Shanghai, China). Lipofectamine RNAiMAX Reagent was purchased from Thermo Fisher Scientific. The western diet (cat. no. TP28640) was purchased from Trophic Animal Feed High‐Tech Co., Ltd. (China), and oxidized low‐density lipoprotein (ox‐LDL; cat. no. YB‐0010) was purchased from Yiyuan Biotech (Guangzhou, China).

The sections (10-μm) were incubated in blocking solution with primary antibodies: anti-nestin (1:400; MAB353, Millipore, Billerica, MA, USA) as a marker of neural stem cells (NSCs) and neural progenitor cells (NPCs), anti-glial fibrillary acidic protein (GFAP) (1:500; No. 3670, Cell Signaling Technology, MA, United States) as a marker of actively dividing astrocytes, anti-neurofilament 200 (NF-200; 1:2000; Sigma) to label neuronal axons, anti-NeuN (1:800; MAB377; Millipore) as a marker of mature neurons, anti-doublecortin (DCX; 1:1000, 4604S, Cell Signaling Technology), a microtubule-associated protein primarily expressed by migratory immature neurons and neuronal neural progenitors, is widely used as a marker of adult neurogenesis, anti-CD68 (Cell Signaling Technology) as a marker of macrophages. Alexa Fluor 594-conjugated or Alex Fluor 488-conjugated secondary antibodies were used for detection. Nuclei were counterstained with 25 μg/mL DAPI (Sigma-Aldrich). Images were taken by a fluorescence microscope (Eclipse 80i, Nikon) equipped with a high-resolution color digital camera (Digital Sight US-U2, Nikon) The results were analyzed using Image-Pro Plus software (Media Cybernetics, Inc.).

Tissues were rinsed with 1 × PBS thrice at room temperature. Blocking was done with 1% BSA (NEB) and 0.1% Tween-20 in 1 × PBS for 1 h at room temperature. Tissues were stained at 4 °C overnight using the following antibodies diluted in blocking solution: anti-LUM (Abcam, ab168384; clone EPR11380(B); Lot GR121948-4; 1:75), anti-MMP2 (Abcam, ab97779; Lot GR3448382-1; 1:200), anti-α-SMA (Abcam, ab7817; clone 1A4; Lot 1009584-11;1:600), and anti-PDGFA (Santa Cruz Biotechnology, sc-9974; clone E-10; Lot C0222; 1:600). PDPN was detected using AF488-conjugated primary antibody (BioLegend, 337005; clone NC-08; Lot B360564; 1:75). Secondary antibody staining was then carried out for 1 h at room temperate using anti-mouse AF594 (ThermoFisher, A11005; Lot 2538976; 1:1000) and anti-rabbit AF488 (ThermoFisher, A11008; Lot 2557379; 1:1000). Finally, samples were stained with anti-CD68 (Cell Signaling Technology, #79594; clone D4B9C; Lot 779594S; 1:50) overnight at 4 °C. After washing with 1 × PBS three times, tissues were counterstained with DAPI (Sigma) before mounting (Vectashield, H-1700-10).

The brain sections were incubated with 5% bovine serum albumin (A3294-10G, Sigma-Aldrich, Missouri, USA) for 1 h at 25 °C. After washing the blocking buffer, the sections were incubated with primary antibodies including anti-CD68, anti-NeuN, and anti-GFAP antibodies (#97778, #94403, #12389, Cell Signaling Technology, Massachusetts, USA, respectively), at 4 °C for 12 h. Then, the sections were washed in PBS and incubated with secondary antibodies (goat anti-mouse IgG H&L Alexa 488, Alexa 555 or goat anti-rabbit IgG H&L Alexa 488, Alexa 555, Abcam, Cambridge, UK) for 2 h at 25 °C. Finally, the sections were washed in PBS and mounted on slides using the Fluoroshield mounting medium with DAPI (ab104139, Abcam). Fluorescence images of the brain tissue were obtained using a fluorescence microscope (Ni-U, Nikon, Tokyo, Japan), and tissue images were merged using ImageJ software (NIH, Maryland, USA).

Western blot was performed according to standard procedure. The treated cells were lysed with ice‐cold RIPA buffer containing both protease and phosphatase inhibitors. The cytoplasmic and nuclear extracts from cells were separated using nuclear and cytoplasmic Extraction kits (#78833; Thermo Fisher Scientific). Protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific). Primary antibodies including anti‐β‐actin (#4790), anti‐FABP4, anti‐CD68, anti‐PCNA (#13110), anti‐p21 (#2974), anti‐Cyclin B1 (#12231), anti‐NF‐κB/p65 (#6956), anti‐Histone H3 (#4499), anti‐FLAG tag (#8146), anti‐ubiquitin antibodies (#3933), and FAK antibody sample kit (#9330) were all purchased from cell signaling technology (Beverly, USA). Anti‐March5 antibody was purchased from ProteinTech (#12213‐1‐AP). Anti‐human USP30 antibody was purchased from Abcam (#ab235299). All bands were visualized by enhanced chemiluminescence and quantified by Image J analysis software.