Eight well glass bottom slides

Eight-well glass bottom µ-slides (Ibidi, Munich, Germany) were coated with decorin (100 μg/mL), endostatin (10 μg/mL), endorepellin (10 μg/mL), perlecan (25 μg/mL), or type I collagen-H (100 μg/mL) (1 h, room temperature) and then blocked with 5 mg/mL of heat-denatured bovine serum albumin (1 h, room temperature). Control coverslips remained uncoated and were then blocked. After washing the coated wells three times with phosphate-buffered saline (PBS: 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 2.7 mM KCl and 137 mM NaCl, pH 7.4), washed platelets (150 μL of 1 × 10⁸/mL) in Hepes buffer pH 7.45 were allowed to adhere for 45 min at 37 °C; then, they washed three times with PBS and fixed with 10% formalin solution (Sigma). Subsequently, the fixed cells were washed three times with PBS and stained with the dye DiOC₆ (2 μM). For wells from each donor, 3 representative microscopic images per coating were taken. Fluorescence images of adherent platelets were captured with the 20× objective lens of a confocal Ti2 microscope (Nikon, Surbiton, UK).
Image analysis was performed by counting the total number of adhered platelets in ImageJ. The extent of platelet spreading was by scoring (visual inspection of morphology) the platelets forming filopodia or lamellipodia, or the platelets with a fully spread shape.

NSC-34 control and MYO9A-depleted cells were seeded onto eight well glass bottom µ-slides (Ibidi) and transfected with the coding sequence of the TrkA receptor as in the recycling assay. After 24 h, a ROCK inhibitor (InSolution™ Y-27632—Calbiochem), was applied at 3nM in serum-free growth medium over-night and NucRed Live 647 ReadyProbes reagent (Invitrogen) was applied for visualization of nuclei. Cells were imaged using a Nikon A1R laser scanning confocal microscope over a period of ten minutes per cell, with z-stacks obtained. Resulting image series were then analysed using Imaris, with automatic detection of TrkA positive vesicles based on thresholding that was manually edited. Vesicles were then tracked by the software over-time and in 3D space to provide an output for speed the vesicles travelled, 10 cells were analysed per condition over three experimental repeats.