Ab134131

The methodology here refers to the published literature [30 (link)]. Tissues including the thrombus and surrounding vein walls were removed after sacrifice and fixed in 4% formaldehyde, dehydrated through a concentration gradient of ethanol, transparent in xylene, and embedded in paraffin. Thin tissue sections (5 μm thick) harvested on day 2 post-modeling underwent stain with hematoxylin-eosin (HE) and were observed under a light microscope. For immunohistochemical staining, after antigen retrieval by microwave heating in citrate buffer (Beyotime, P0081), sections were blocked with 5% bovine serum albumin (BioFroxx, 4240GR005) before anti-CD41 (Abcam, ab134131) incubation at 4°C overnight. Subsequently, the goat anti-rat IgG secondary antibody (ZGBBT, ZB-2301) was applied for 1 hour at room temperature (RT) followed by the use of diaminobenzidine substrate to visualize the stain sections. The presence of brown spots in the image showed positive staining. Antibodies against CD62P (Proteintech, 60332) and CD41 were employed for immunofluorescence double-staining. The slides were incubated with Dylight 488-conjugated goat anti-rat IgG secondary antibodies (Abbkine, A23220) or Dylight 594-conjugated goat anti-mouse IgG secondary antibodies (Abbkine, A23410). 5 to 8 randomly unrepeated field areas were selected to view under ×400 magnification in an optical microscope.

Total protein was extracted from tissues or RAW264.7 cells using Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (R0010; Solarbio). After SDS‐PAGE separation and membrane transfer, immunoblots were obtained by using the following primary rabbit antibodies: GAPDH (#5174, 1:1000, Rabbit, Cell Signaling Technology), TLR4 (ab13556, 1:500; Abcam Inc.), phosphorylated‐ERK1/2 (p‐ERK1/2) (ab17942, 1:1000; Abcam), ERK1/2 (ab17942, 1:1000; Abcam), KLF4 (ab129473, 1:1000; Abcam) and ITGA2B (ab134131, 1:2000; Abcam). The immunoblots were visualized with secondary goat anti‐rabbit antibody to immunoglobulin G (ab150077, 1:1000; Abcam) and enhanced chemiluminescence, and analysed by Image J software. The relative expression of protein was expressed as the ratio of grey value of the target band to that of internal reference (GAPDH).

For Wright-Giemsa staining, cultured cells were centrifuged (at 400 × g for 2 minute) onto glass slides, and cytospin specimens were subjected to Wright–Giemsa staining, and examined by light microscopy (microscope, Leica DM2000, N PLAN ×100/1.25 Oil, Leica Microsystems GmbH, Wetzlar, Germany; Digital camera, LONGBASE 1010, LONGBASE, Qingdao, China). For immunofluorescence staining, cultured cells were stained with anti-CD41 antibody (1:200; ab134131, Abcam, Cambridge, UK), Alexa Fluor 488 secondary antibody (1:2000; A21206, Invitrogen, Carlsbad, CA, USA), and Flouroshield containing DAPI (ab104139, Abcam, Cambridge, UK), and were dropped onto glass slides, and examined by immunofluorescence microscopy (EVOS FL Auto 2, Invitrogen, Carlsbad, CA, USA).

The degree of platelet adhesion was determined after incubation of test specimens 0.25 cm² in size with 300 μl of PRP for 1 h at 37°C. In order to remove non-adherent platelets, the products were washed in phosphate-buffered saline (PBS; pH 7.4), then fixed in 4% paraformaldehyde solution for 10 min. Next, the specimens were incubated with rabbit antibodies to CD41 (ab134131; Abcam, UK) and mouse antibodies to CD62P (ab54427; Abcam, UK) for 12 h at 4°C. After that, the matrices were washed with PBS with the addition of 0.1% Tween 20. Then, the specimens were incubated for 1 h at room temperature with goat secondary antibodies to rabbit IgG conjugated with Alexa Fluor 488 (A11034; Thermo Fisher Scientific, USA) and goat antibodies to mouse IgG conjugated with Alexa Fluor 555 (A31570; Thermo Fisher Scientific, USA). The sections were repeatedly washed with PBS with the addition of 0.1% Tween 20. A confocal microscope (LSM700; Carl Zeiss, Germany) was used to analyze the products.

Cryostat sections from macroscopic blood clots, obtained by tubing loop and embedded in Tissue-Tek O.C.T (Sakura Finetek, Torrance, CA, USA), were fixed in 4% para-formaldehyde and 50% ethanol before analysis. Slides were stained for platelets with an anti-CD41 antibody (rabbit anti-human 1:250, ab134131, Abcam, Cambridge, United Kingdom), for thrombin (mouse anti-human 1:200, ab17199, Abcam), for cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and for cells with cell tracker red (Red CMTPX Dye, C34552, Thermo-Fischer Scientific). Four channel images were sequentially collected by a Cell observer Spinning Disk microscope (Zeiss, Oberkochen, Germany), equipped with a Plan-Neofluar 40×/1.30 oil objective and excitation laser lines of 405, 488, 561 and 635 nm, and emission channels at 460/80, 520/35, 617/73 and 685/40 nm.

Platelet lysates that contain 20 μg proteins were electrophoresed on 6% polyacrylamide gels for 45 minutes at 200 V. Proteins were transferred from gels onto 0.45μm polyvinylidene difluoride (PVDF) membranes (IPVH00010, Immobilon-P) for 1 hour at 100 V. Membranes were blocked with 5% w/v non-fat powdered milk in Tris-buffered saline-Tween (TBS-T; 137 mM NaCl, 20 mM Tris, 0.1% Tween-20, pH 7.6) for 1 hour at room temperature, incubated overnight with agitation at 4 °C with 1:10,000 anti-talin antibody (clone 8D4; T3287, Sigma) or 1:5000 anti-CD41 antibody (ab134131, Abcam) and washed three times with TBS-T. To detect the primary antibody, membranes were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (7074 S, Cell Signaling Technology) or anti-mouse IgG (7076 S, Cell Signaling Technology), again washed three times with TBS-T. To visualise the blot, membranes were exposed to Super Signal Chemiluminescent Substrate (34077, Thermo Scientific) for 5 minutes. The blots were exposed to X-ray films (Amersham Hyperfilm ECL, GE Healthcare) and developed using an OptiMax X-ray film processor (Protec Medizintechnik).