STED super-resolution microscopy 3D imaging of talin and αIIb localization in platelets. (A-J) SHARPIN^fl/fl Pf4-Cre⁺ and SHARPIN^fl/fl Cre⁻ platelets were allowed to spread on fibrinogen-coated coverslips for 30 minutes and then stained with primary antibodies against αIIb and talin and secondary Alexa-594 goat anti-rat and Atto 647N goat anti-mouse antibodies, respectively. Sixteen 0.1-μm sections that spanned the entire platelet were acquired with a Leica SP8 confocal STED microscope with white light laser, equipped with a ×100 STED oil objective. Images were processed using Leica LASX software and Photoshop, with linear adjustments applied similarly. (A-B) Front view of sample platelets from SHARPIN^fl/fl Pf4-Cre⁻ (A) and SHARPIN^fl/fl Pf4-Cre⁺ (B) mice. Arrowheads indicate peripheral regions of αIIb and talin association, pseudocolored green and red, respectively. (C-D) Quantification of αIIb and talin association, as indicated by Pearson’s colocalization (C) and colocalization overlap analyses (D). (E-F) Clipping views of SHARPIN^fl/fl Pf4-Cre⁻ (E) and SHARPIN^fl/fl Pf4-Cre⁺ (F) fibrinogen-adherent platelets showing central apical-dorsal “pillars” of αIIb and talin that traversed the platelet body and appeared as an organized assembly in 3D space. Arrows denote relative maximum heights of αIIb and talin. (G-J) Quantification of apical-dorsal pillars. Platelet αIIb staining was used to set a stringent threshold for pillar discrimination and applied identically to images of SHARPIN^fl/fl Pf4-Cre⁻ and SHARPIN^fl/fl Cre⁺ platelets. (G) Maximal pillar height. **P < .01. (H) Average pillar volume. (I) Average pillar surface area. (J) Average pillar diameter. SHARPIN^fl/fl Pf4-Cre⁻ platelets showed significantly higher pillar volume and surface area than their wild-type counterparts (H-I), but no difference in column diameter was found (J). Approximately 25 to 40 cells were analyzed for each group. Data represent the sum of 4 individual experiments. n.s., not significant; *P < .05.