Ab6632

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.

Following whole blood perfusion experiments, adherent platelets were fixed with 10% neutral buffered formalin for 30 minutes. The slides were blocked with 1% BSA for 1 hour prior to the addition of 1:1000 dilution anti‐human GpIbα (AF4067; R&D Systems, Abingdon) or anti‐P‐selectin (ab6632; abcam) overnight at 4°C. Slides were washed and anti‐sheep‐FITC (F5137; Sigma) or anti‐mouse Alexa‐647 (115‐606‐008; Jackson ImmunoResearch) conjugated secondary antibodies added for 1 hour at 24°C prior to washing. Images were then exported to ImageJ1.35 for analysis. Overall fluorescence was measured as Mean Gray area. Images were subjected to thresholding in order to obtain fluorescent surface area.
For original data, please contact rwf10@cam.ac.uk.

A protein extraction kit (Ding Guo, China) was used to extract total cell protein. The extracted proteins from platelets, Pex and PexD were boiled for 5 min and separated by SDS-PAGE. Coomassie blue staining was used to visualize proteins. Proteins were prepared for western blotting as previously stated. Following gel electrophoresis, proteins were transferred to polyvinylidene fluoride membranes (Bio-Rad). After 2 h of blocking in 5% skim milk, the membranes were incubated with anti-TSG101 (Abcam, ab125011), anti-CD9 (Abcam, ab92726), anti-CD63 (Abcam, ab217345), anti-CD61 (Abcam, ab119992), anti-CD41 (Abcam, ab63983) or anti-P-selectin (Abcam, ab6632). The antibodies were added at a dilution of 1:1000 and incubated at 4 °C overnight, and the blots were then incubated with 5% skim milk for 2 h. Then, the membranes and an appropriate secondary antibody (1:10,000) were incubated at room temperature for 1 h.