FITC-Fg transits through Rab4- and Rab11-positive compartments after internalization. (A) Rab4- and Rab11-containing compartments are present in human platelets. Washed human platelets (5 × 108/mL) were fixed with 2% paraformaldehyde, washed, and allowed to adhere to poly-d-lysine–coated coverslips. Immunofluorescence staining was done as described in “Methods.” Platelets were incubated with anti-Rab4 mouse monoclonal antibody and anti-Rab11 rabbit polyclonal antibody, and then with Alexa 568 conjugated goat anti-mouse IgG or Alexa 488 conjugated anti-rabbit IgG, respectively. Images were taken with a Nikon A1R confocal microscope (60X/1.49 NA DIC N2 oil) and digitally magnified ×30. Scale bar represents 1 µm. (B) Internalized FITC-Fg goes through Rab4- and Rab11-positive compartments in WT mouse platelets. Washed WT mouse platelets (5 × 108/mL) in HEPES Tyrode buffer containing 1 mM Ca2+ were incubated with FITC-Fg (0.05 mg/mL) for the indicated times and then fixed with 2% paraformaldehyde at room temperature. After washing with PBS, the fixed platelets (5 × 107/mL) were allowed to adhere to poly-d-lysine–coated coverslips. Immunofluorescence staining was performed as described in “Methods” using anti-Rab4 rabbit polyclonal antibody or anti-Rab11 rabbit polyclonal antibody followed by TRITC-conjugated goat anti-rabbit secondary antibody. Images were taken with a Nikon A1R confocal microscope (60X/1.49 NA DIC N2 oil) and digitally magnified ×15. Scale bar represents 1 µm.
