Af6990

Cross-sections of carotid arteries were used for confocal microscopic imaging. Platelets were stained with antibodies against CD42d (AF6990, R&D, USA), CD41 (553847, BD, USA) and PF4 (ab282111, Abcam, USA). Monocytes/macrophages were stained with rat anti-mouse F4/80 antibody (MAB5580, R&D, USA). Neutrophils were stained with Ly6G antibody (sc-52515, SANTA CRUZ, USA). Endothelial cells were stained with rabbit anti-mouse CD31 (ab28364, Abcam, USA). Podoplanin expression was determined with Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA). Cytoskeletal proteins were determined with phalloidin antibody (CA1610, Solarbio, China). Secondary antibodies Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated donkey anti-rabbit IgG, and Alexa Fluor 647-conjugated goat anti-Syrian hamster IgG were all from Abcam. Donkey anti-sheep IgG NorthernLights™ NL557-conjugated antibody was purchased from R&D. Tissues were counterstained with DAPI before being mounted and examined by confocal microscopy, an Olympus multiphoton laser scanning microscope with high S/N ratio objectives and suppressed autofluorescence, which enables advanced deeper imaging with high resolution (FV1000MPE and FV3000MPE Olympus, Japan).

IHC was performed as previously described [18 (link)]. In brief, the mice were anesthetized using 4% chloral hydrate (350 mg/kg) and perfused with PBS, and then 4% paraformaldehyde was injected through the left cardiac ventricle. The brains were extracted and fixed in 4% paraformaldehyde at 4 °C for 24 h, immersed in phosphate buffer containing 20% or 30% sucrose, and sliced into 10-mm-thick sections using a cryostat. The sections were rinsed with PBS for 15 min, blocked with 1% bovine serum albumin (BSA) in 0.3% Triton X-100 for 1 h, and then incubated overnight with mouse anti-CD42d (1:100, AF6990, R&D, USA), mouse anti-TNF-α (1:100, ab1793, Abcam, USA), and rabbit anti-GFAP (1:500, AB5804, Millipore, USA) antibodies. After being washed again with PBS, the sections were incubated with Alexa Fluor 488 donkey anti-sheep IgG(H + L) (1:200, A-11015, Invitrogen, USA), Alexa Fluor 488 sheep anti-mouse IgG (H + L) (1:500, AB150113, Abcam, USA), and Alexa Fluor 647 donkey anti-rabbit IgG(H + L) (1:500, AB150063, Abcam, USA) secondary antibodies for 1 h at room temperature in the dark. Then, the sections were counterstained with DAPI (1: 10,000, D9564, Sigma), dried, and mounted on coverslips. Images were obtained under a confocal microscope (LSM 710, Carl Zeiss Co. Ltd., Oberkochen, Germany).

En face whole-mount immunostaining was performed on the luminal side of common carotid arteries as described previously 39 (link). In brief, dissected arteries were fixed in 4% paraformaldehyde (PFA) for 4 h and washed with 0.3% PBSTX (0.3% Triton X-100 in PBS) for 4 times (15 min/time) at room temperature (RT). After washing, samples were blocked with 3% PBSMT (3% non-fat milk in PBSTX) at 4 °C overnight and then incubated with a primary antibody (in 3% PBSMT) at 4 °C overnight. After washing with 0.3% PBSTX for 4 times (30 min/time), the samples were incubated with a fluorescent-conjugated secondary antibody (1 μg/ml in 0.3% PBSTX) at 4 °C overnight. After washing with 0.3% PBSTX for 4 times (30 min/time), the samples were incubated 15min with DAPI at room temperature, then washed 2 times and were fixed with 1% PFA for 3 min and washed with 0.3% PBSTX prior to mounting with cover slides and an anti-fading agent (0100-01, Southern Biotech, USA). Antibodies used in the en face immunostaining: rabbit anti-mouse CD31 (ab28364, Abcam, USA), sheep anti-mouse CD42d (AF6990, RD, USA), rat anti-mouse F4/80 (MAB5580, R&D, USA), Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA).