Immunofluorescence imaging and localization of CFH within normal platelets. Washed normal human platelets were fixed, permeabilized, and incubated with pairs of mouse monoclonal and rabbit polyclonal antibodies, followed by labeling with secondary antibodies tagged with Alexa Fluor 568 or Cy3 (red), or Alexa Fluor 488 (green). White bars with the corresponding microns are indicated. All images were deconvolved. (A-B) Rendered 3-dimensional images. (C-D) Representative midplatelet Z-slices. (A) Staining of intact platelets for actin reveals the plate-like shape of resting cells in both top (left) and perpendicular (right) views, whereas (B) tubulin-staining microtubule rings (red) surrounding VWF-containing α-granules (green) confirm the nonactivated state. (C-D) CFH labeled with Alexa Fluor 488 (green) is shown in the middle panels; right panels, the corresponding merged left and middle panels. Costaining of CFH (green) with VWF (C, red) or fibrinogen (D, red) confirms the presence of CFH throughout platelets and does not indicate specific colocalization with either α-granule marker. Results of whole-platelet colocalization analysis are presented in Table 1.