Ab63983

The protein expression of CD62-P (P-selectin), CD41 and CD44 was evaluated by indirect immunofluorescence and confocal microscopy analysis. Paraffin-embedded sections were permeabilized in a phosphate-buffered saline (PBS) with 0.05% TWEEN-20 for 5 min, washed in PBS and then blocked with a 2% goat serum in PBS for 1 h at room temperature (RT). The sections were incubated overnight (ON) in a humidified chamber at 4 °C with a primary antibody against CD62-P (1:500) (ab6632, Abcam, Cambridge, UK), CD41 (1:100) (ab63983, Abcam, Cambridge, UK) or CD44 (1:500) (ab6124, Abcam, Cambridge, UK) following incubation for 1 h with the appropriate secondary antibody (Alexa Fluor 488 goat anti-mouse, 1:200, A32723, Thermo Fisher Scientific; Alexa Fluor 555 goat anti-mouse, 1:200, A32727, Thermo Fisher Scientific). The sections were counterstained with To-Pro (Thermo Fisher Scientific), mounted in Fluoromount (Biomeda Corp, Foster City, CA, USA) and sealed with nail varnish. Negative controls were performed by omitting the primary antibodies. Specific fluorescence was acquired by a Leica TCS SP8 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using a 630× objective lense and 5× optical zoom.

Three sections (100 µm intervals) were prepared from the middle portion of the thrombus and immunohistochemically stained. For the immunological staining of fibrin(ogen), anti-fibrin(ogen) antibody (1:200, ab63983; Abcam, Cambridge, MA, USA) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) were used. For Ly-6G, Alexa Fluor 647–conjugated anti-mouse Ly-6G (1:400, BD127626; BioLegend, San Diego, CA, USA) was used. For CD42b, anti-CD42b antibody (1:1000, ab104704; Abcam) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific) were used. For Cit-H3, anti-Cit-H3 antibody (1:250, ab5103; Abcam, Cambridge, MA, USA) and Alexa Fluor 488–conjugated secondary antibody (Thermo Fisher Scientific) were used. Nuclei were labeled using 4’, 6-diamidino-2-phenylindole (excitation wavelength: 360 nm, fluorescence wavelength 461 nm, 62248; Thermo Fisher Scientific) or SYTOX Green (excitation wavelength: 504 nm, fluorescence wavelength 523 nm, 33025; Thermo Fisher Scientific). Sections were examined using an LSM 510 META confocal microscope (Carl Zeiss, Jena, Germany). Non-immune rabbit immunoglobulin G and polyclonal isotype control were used as negative controls of fibrin(ogen), CD42b, and Cit-H3. Positive staining was evaluated using ImageJ software v1.50i (https://imagej.nih.gov/ij/index.html, accessed on 20 October 2021).

The freshly harvested tissues were formaldehyde-fixed prior to freezing and were sectioned using a cryostat (Leika CM1850; Leica Microsystems GmbH, Nussloch, Germany) to 4 μm. The tissue sections were then incubated with rabbit anti-CD41 polyclonal antibody (1:100; ab63983), mouse anti-CD31 monoclonal antibody (1:100; ab24590) and/or mouse anti-VEGF monoclonal antibody (1:200; ab1316), which were all purchased from Abcam, overnight at 4°C. The tissue sections were then incubated with a secondary goat anti-rabbit fluorescein isothiocyanate (111-095-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and goat anti-mouse Rhodamine (115-295-003; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 1 h. The nuclei were counterstained with diamidinophenylindole (Sigma-Aldrich, St. Louis, MO, USA) and fluorescent images were captured using microscopy (BX51; Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus software for colocalization.

Western blotting for the EV markers CD63, CD9, and HSP70 (EXOAB-KIT-1, System Biosciences), TSG101 (ab83, Abcam), and ALIX (ab117600, Abcam) was undertaken as previously described by Lötvall et al. (63 (link)). The purity of EV samples was tested for contaminating intracellular organelles by ATP5A (mitochondria) (15H4C4, Abcam), nuclear fractions by histones (histone H3) (D1H2/4499P, Cell signaling), and platelets by CD41 (ab63983, Abcam). Briefly, protein quantity in EVs was detected by lysing EV pellets in CelLytic MT Cell LysisReagent or RIPPA buffer (Cell Signaling) with added protease inhibitors (Roche Complete). To ensure equal loading, protein concentration was measured by bicinchoninic acid assay (Thermo Scientific), and 10–40 μg protein was loaded per well. SDS page samples were separated on 4%–12% bis-tris gradient gels (Life Technologies) under nonreducing conditions. Separated samples were transferred to PVDF or nitrocellulose membranes, and nonspecific binding was blocked with 5% milk in PBS-Tween. Membranes were incubated with antibodies overnight, and membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase. Membranes were washed again before incubation with enhanced chemiluminescence substrate (Pierce ECL, ThermoFisher Scientific) and imaged on a Bio-Rad ChemiDoc MP Imaging system.