Anti cd68

All samples were stained as previously described by our laboratory(Hill et al. 2018a (link); Hill et al. 2018b (link)). Antibodies used were anti-Ki67 (1:500, Abcam, Cambridge, UK; Cat# ab15580 Lot# GR264777 RRID:AB_443209), anti-DCX (1:1000, Millipore, Burlington, MA, USA; Cat# AB2253 Lot# 2828588 RRID:AB_1586992), anti-BrdU (1:200, Abcam, Cat# ab6326 Lot# GR267766–1 RRID:AB_305426), anti-IBA-1 (1:500, Wako Chemicals, Richmond, VA, USA; Cat# 019–19741 Lot#WDK2121 RRID:AB_839504), and anti-CD68 (1:1000, Bio-Rad Laboratories, Hercules, CA, USA; Cat# MLA1957 Lot#1708 RRID:AB_322219). Secondary antibodies used were Alexa-488® goat anti-guinea pig (Thermo Fisher Scientific, Waltham, MA, USA; Cat# A-11073 Lot#1841755 RRID:AB_2534117), Alexa-594® donkey anti-rat (Thermo Fisher Scientific; Cat# A-21209 Lot#45081A RRID:AB_2535795), Alexa-488® donkey anti-rabbit (Thermo Fisher Scientific; Cat# A-21206 Lot#1608521 RRID:AB_2535792). All secondary antibodies were used at a dilution of 1:400. Imaging for in vivo quantification was performed using a CoolSNAP EZ CCD camera (Photometrics, Tucson, AZ, USA) coupled to a Nikon i80 Eclipse (Nikon Instruments Inc., Melville, NY, USA). Confocal images were taken on a Nikon A1R confocal microscope (Nikon).

Two hours or five days after the surgery, the mice were perfused with PBS and the brains were removed and fixed in 4% PFA for 24 h before being placed into 30% sucrose for cryoprotection. Brains were sectioned coronally at 20 μm on a cryostat and mounted on glass slides. Frozen mouse sections (n=8 sections/animal, 80 μm interval, covering most of sections with obvious FLsAβ₄₂ signal) were blocked with 8% goat serum and 3% BSA in PBS containing 0.3% Triton X-100, and then incubated with anti-Iba-1 (1:200; Wako), anti-CD68 (1:200; Bio-rad), and anti-LAMP-1 (1D4B, 1:100; DSHB) antibodies overnight at 4 °C. After washing with PBS, sections were incubated with suitable Alexa Fluor second antibodies for 1 h at room temperature and nuclei were visualized by TO Pro-3 staining. Images of the injected frontal cortex with FLsAβ₄₂ signal were obtained as a z-series stack (1 μm) using a Zeiss LSM 510 confocal microscope. The imaging and quantitation were performed by the investigator who were blinded to the experimental groups. The mean intensities of microinjected FLsAβ42 on each section at 2 h and 5 days after surgery were analyzed.

CD19-reporter mouse hearts were fixed and processed as previously described [3 , 13 ]. Heart sections were stained with the primary antibodies anti-CD68 (Bio-Rad; Cat#MCA1957T; 1:400) and anti-CD31 (R&D Systems; Cat#AF3628; 1:15), overnight at 4°C, followed by incubation with donkey anti-rat Alexa Fluor 488 (ThermoFisher; Cat#A-21208; 1:200) and NL637 (R&D Systems; Cat#NL002; NorthernLights anti-goat IgG-NL637; 1:200) for 1–4 h at room temperature. DAPI was used to stain the nucleus. Fluorescence was visualized using a Zeiss LSM 880 Confocal microscope at the Washington University Center for Cellular Imaging (WUCCI).

Formalin fixed, paraffin embedded sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in Diva Decloaker (pH 6.2, Biocare Medical). They were blocked in 10% donkey serum for 1 h to prevent nonspecific binding. The sections were then incubated overnight at 4°C in primary antibody (anti-CCR2, Novus Biologicals, 1:100 and anti-CD68, Biorad, 1:100) or control IgG (anti-rabbit and anti-mouse IgG, Novus Biologicals, 1:400). Anti-rabbit and anti-mouse secondary antibodies were applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in phosphate-buffered saline (PBS), mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Leica fluorescent microscope system. In addition, hematoxylin and eosin (H&E) stains were performed on serial sections to analyze morphology and severity of the AAA tissues. The infiltrated inflammatory cells were counted by a clinical pathologist at 3 randomly selected 20x region-of-interest of AAA and sham-control tissue slides (n=4/group). Pentachrome stain was also carried out to characterize the collagen content of AAA and sham-control tissues.

Brain tissues were homogenized in lysis buffer (150mM NaCl, 10mM NaH2PO4, 1mM EDTA, 1% TritonX100, 0.5% SDS) with protease inhibitor cocktail and phosphatase inhibitor (Sigma). Equivalent amounts of protein were analyzed by 4-15%Tris-HCl gel electrophoresis (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membranes and probed with antibodies. Visualization was enabled using enhanced chemiluminescence (GE Healthcare Pharmacia). The following primary antibodies (dilutions) were used: anti-CD68 (1:1000, BioRad), anti-CD11b (1:1000, Abcam), anti-Iba1 (1:1000, Millipore), anti-Phopspho-p38MAPK (1:1000, Cell Signaling), anti-p38MAPK (1:1000, Cell Signaling) and β-actin (1:2000, Cell Signaling). Secondary antibodies were HRP-conjugated anti-rabbit or anti-mouse antibody (1:1000, GE Healthcare).