Microscopic detection of ADAP association with talin and kindlin-3 in human platelets. Washed human platelets were stimulated with SFLLRN, plated on fibrinogen-coated coverslips for 1 hour, and processed as described in Methods. (A-B) Permeabilized platelets were stained with antibodies against ADAP (red) and talin (green) (A), kindlin-3 (green) (B), or control immunoglobulin G (IgG) antibodies. Cells were counterstained with rhodamine-phalloidin to label F-actin (blue). Arrows indicate co-localization of all 3 stained proteins. (C-D) PLA to detect interactions between ADAP and talin (C) or ADAP and kindlin-3 (D). Specific PLA signals appear as bright red dots when ADAP-talin (C) and ADAP-kindlin (D) primary antibodies were used (arrows), but not when control rabbit (Rb-) or murine (m-) IgG antibodies were used. Platelets were counterstained with FITC-phalloidin (green). (E-F) Quantification of average proximity PLA signals/platelet between ADAP and talin (E) or ADAP and kindlin-3 (F) (*P < .01). Results represent the mean ± standard error of the mean of 3 experiments.