Figure 5: SIM and STED Imaging of Collagen Stained With Not Cross-Linked GPVI-Fc (GPVI-Fc*IgG, GPVI-Fc*Fab2) and Cross-Linked GPVI-Fc (GPVI-Fc*IgG-XL, GPVI-Fc*Fab2-XL)

GPVI-Fc was incubated with Alexa Fluor 488-conjugated anti–human-Fc IgG or anti–human-Fc Fab2 antibodies in either a 10:1 molar ratio (not cross-linked GPVI-Fc) or a 1:1 molar ratio (cross-linked GPVI-Fc). The mixtures were then incubated with collagen coated glass coverslips. (A) SIM imaging. The SIM micrographs are maximum intensity projections of 0.15 μm z sections (total z 1.5 μm). The image is representative of 5 others from different experiments. (B) Stimulated emission depletion (STED) microscopy imaging. Collagen coated glass coverslips were stained simultaneously with AlexaFluor594-labeled anti-collagen type I and type III antibodies (red) in addition to GPVI-Fc, which was either labeled or cross-linked with anti–human-Fc antibodies. Yellow indicates co-localization of cross-linked GPVI-Fc with anti-collagen antibodies. Abbreviations as in Figures 1, 2, and 4.