Vb 7000

Cells were grown on glass coverslips, exposed (or not) to X-ray or carbon-ion beam irradiation, and then stained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), as described previously [20] (link). Confocal images were collected using a BX51 microscope (Olympus) equipped with a CCD camera (VB-7000; Keyence). Apoptosis was determined based on the morphology of the nuclei, including the presence of apoptotic bodies, nuclear condensation and fragmentation [21] (link). Cells containing nuclei with two or more distinct lobes were scored as positive for mitotic catastrophe [20] (link), [22] (link). Cells containing nuclei showing senescence-associated heterochromatic foci were scored as positive for senescence [23] (link). The percentages of cells undergoing apoptosis, mitotic catastrophe or senescence were quantified by counting at least 300 cells for each experimental condition.

Fixed platelets on the cp-Ti plates were directly subjected to treatment with primary antibodies, such as mouse monoclonal anti-CD62P antibody (1:20 dilution; BioLegend, San Diego, CA, USA), rabbit polyclonal anti-PDGF-B (1:200 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-TGFβ1 (1:200 dilution; Santa Cruz Biotechnology), rabbit monoclonal anti-PPARγ (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-fibrin antibodies (1:400 dilution; GeneTex, Inc., Irvine, CA, USA) overnight at 4 °C. In this study, based on our preliminary observations that our routine blocking or washing with Tween-20-containing PBS also visualized intra-platelet binding of the antibodies we used, we did not perform blocking or use such a detergent-containing PBS in the main experiments in this study.
The samples were then probed with the corresponding secondary antibodies or non-immunized rabbit IgG (Life Technologies Corporation, Carlsbad, CA, USA) as an isotype control for 60 min at ambient temperature in the dark. The samples were finally mounted using an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA) and examined under a fluorescence microscope (ECLIPSE 80i; Nikon) connected with a cooled CCD camera (VB-7000; Keyence) [23 (link)].

C57BL/6J male mice were used after being sacrificed by anesthesia with diethyl ether. Mice were transcardially perfused, initially with PBS and then with 4% PFA in PBS. Tissues were dissected out, postfixed in 4% PFA at 4°C for 5 h, and cryoprotected by immersion in 15% sucrose in PBS overnight at 4°C. After embedding in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan), tissues were frozen in dry ice powder, and sectioned at a thickness of 15 µm using a cryostat (CM1850; Leica Microsystems, Frankfurt, Germany) at −18°C. Sections were air-dried for 1 h, and rinsed three times in PBS. After blocking with 5% donkey normal serum (Vector, Burlingame, CA, USA) in PBS, sections were reacted with specific primary antibodies at 4°C overnight, rinsed in PBS, reacted with the appropriate secondary antibody at room temperature for 1 h, and again rinsed in PBS. Immunoreacted sections were mounted with Vectorshield (Vector) mounting medium, and observed using a microscope (BX51, Olympus, Tokyo, Japan) equipped with a CCD camera (VB-7000, Keyence, Osaka, Japan). Digital images were processed using Adobe Photoshop 6.0 software.

Immunohistochemistry was performed as previously described [24 (link)]. Briefly, C57BL/6J male mice were deeply anesthetized with a combination of midazolam, medetomidine, and butorphanol tartrate and transcardially perfused with PBS, followed by Zamboni’s fixative. The tissues were dissected, post-fixed in Zamboni’s fixative at 4 °C for 5 h, and cryoprotected by immersion in 15% sucrose in PBS overnight at 4 °C. After embedding in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan), the tissues were frozen and sectioned at a thickness of 15 μm using a cryostat (CM1950, Leica Microsystems, Frankfurt, Germany) at − 18 °C. The sections were air-dried for 1 h and rinsed in PBS three times. After blocking with 5% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS at room temperature for 1 h, the sections were incubated at 4 °C overnight with primary antibodies in immunoreaction buffer (2×PBS containing 0.3% Triton X-100 and 1% BSA). The sections were then washed in PBS, incubated at room temperature for 1 h with the appropriate secondary antibodies in the immunoreaction buffer, and washed again in PBS. Stained sections were mounted in DAPI Fluoromount-G® mounting medium (SouthernBiotech) and observed by a fluorescence microscope (BX51; Olympus) equipped with a CCD camera (VB-7000; Keyence). Researchers analyzing the images were blinded to the experimental conditions.

Immunocytochemistry was performed as previously described [23 (link)]. Briefly, cells were fixed with Zamboni’s fixative (2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.2% picric acid) at room temperature (RT) for 15 min. After washing three times with phosphate-buffered saline (PBS), the cells were permeabilized in PBS containing 0.02% Triton X-100 at RT for 5 min. After blocking with Image-iT FX signal enhancer (Thermo Fisher Scientific, MA, USA) at RT for 60 min, the cells were incubated with primary antibodies at 4 °C overnight, rinsed in PBS, incubated with Alexa-conjugated secondary antibodies at RT for 1 h, and rinsed again in PBS. Immunoreactive cells were mounted in DAPI Fluoromount-G® mounting medium (SouthernBiotech, Birmingham, AL, USA) and observed under a fluorescence microscope (BX51; Olympus, Tokyo, Japan) equipped with a CCD camera (VB-7000; Keyence, Osaka, Japan). Researchers analyzing the images were blinded to the experimental conditions.

Platelets were inoculated onto the surface of cp-Ti plates and incubated for 30 min. Then, the cp-Ti plates were washed with PBS to remove nonadhered platelets, as described earlier, and were further incubated for 2 h with a highly water-soluble tetrazolium dye (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). After incubation, 100 μL of supernatant was collected, and its absorbance was measured at 450 nm.
Alternatively, platelets were fixed with 10% neutralized formalin, microperforated with 0.1% Tween-20-containing PBS (T-PBS) for 1 min, and stained with phalloidin (Cytopainter Phalloidin-iFlour 555 Reagent; Abcam, Cambridge, MA, USA) at ambient temperature in the dark and observed under a fluorescence microscope (ECLIPSE 80i; Nikon, Tokyo, Japan) connected with a cooled CCD camera (VB-7000; Keyence, Osaka, Japan).

The expression of the fluorescent proteins GFP and DsRed was detected using an MZFLIII (Leica, Solms, Germany), MZ16FA (Leica), SZX16 (Olympus, Tokyo, Japan), or VB-S20 fluorescence microscope (Keyence, Osaka, Japan) with a GFP band pass filter (excitation: 440/70 nm, emission: 525/50 nm or excitation: 440/70 nm, emission: 535/550 nm or excitation: 460/80 nm, emission: 495/540 nm), GFP long-pass filter (excitation: 440/70 nm, emission: 510 nm) or DsRed filter (excitation: 525/40 nm, emission: 572 nm or excitation: 530/45 nm, emission: 620/60 nm). Images were captured using a Leica DC200 (Leica), Leica DFC300FX (Leica), DP71 (Olympus), or VB-7000 (Keyence) system.