Cd42b

Retrieved thrombi from thrombectomized IS patients (n = 44) were transferred to saline until fixed in formalin (PanReac AppliChem, Spain) during 24 h. Then, samples were embedded in paraffin by a tissue automatic processor (Tissue-Tek VIP, Sakura, Japan) and paraffin-embedded clot material was cut into 3-μm sections with a rotatory microtome (HM-340E, Microm, Germany). Serial slides from each thrombus were stained with hematoxylin and eosin (PanReac AppliChem, Spain), with Martius Scarlet Blue staining (Atom, UK) and with specific antibodies against platelets (CD42b, Invitrogen, USA), neutrophil elastase (NE, Sigma-Aldrich, USA), and the calcium-binding protein S100A9 (PA5-82145, Invitrogen, USA) as part of the heterodimer calprotectin. Immunostained slides were scanned (Aperio ImageScope, Leica ByoSistems, Germany) and quantified with ImageJ software [17 (link)]. Data are presented as the percentage of positive stained area in the total tissue area.

The 3D‐printed silk bioink was housed in a customized device manufactured using 3D Stereolithography (LSA) printing technology (FormLab). The model was created using Fusion 360 (AutoDesk), and PreForm software was used for slicing (FormLab). The printing was done using a long‐term non‐toxic biocompatible resin (FormLab). A glass window at the bottom ensured possibilities for live‐cell imaging and high‐resolution microscopy. For immunofluorescence imaging samples were fixed in 4% formaldehyde for 20 min, at room temperature. Samples were probed with anti‐CD34 (1:100), anti‐CD61 (1:100), anti‐CD41 (1:100) (Beckman Coulter), CD42b (1:100) (Invitrogen), or anti‐β1‐tubulin (1:200) (Abcam), overnight at 4 °C. Alexa Fluor secondary antibody (1:500) (Invitrogen) have been incubated for 2 h at room temperature. Nuclei were stained with Hoechst 33 258 (Sigma–Aldrich). Samples were imaged by an SP8 confocal laser scanning microscope (Leica). For live imaging, samples were stained with FITC conjugated anti‐CD61 or anti‐CD41 antibodies, or Cytopainter Cell Plasma Membrane Staining Kit and NBD Cholesterol Staining Dye Kit (Abcam). Nuclei were stained with BioTracker NIR694 Nuclear Dye (Sigma‐Aldrich). Isotype controls were used as a negative control to exclude non‐specific background signals. The acquisition parameters were set on the negative controls.