Subcellular localization of HBP by immunofluorescence microscopy.

Indirect immunolocalization of HBP and markers for azurophilic granules and secretory vesicles. Neutrophils were fixed, permeabilized, and stained, as described in “Materials and methods,” before attachment to poly-L-lysine–coated coverslips and recording of images. (A-C) Cells were incubated with a rabbit anti-HBP antibody and a mouse monoclonal antibody against the azurophilic granule marker, CD63. (D-F) Cells were incubated with a rabbit anti-HBP antibody and a mouse monoclonal antibody against the secretory vesicle marker, CD35. Thereafter, the cells were stained with FITC-labeled anti-rabbit and Cy3-labeled anti-mouse secondary antibodies. Staining of HBP (A, D), CD63 (B), and CD35 (E). Corresponding Nomarski images (C, F). Results are representative of more than 5 separate experiments. (Bar = 10 μM). (inset) Specificity of the rabbit anti-HBP antibody was verified by Western blot analysis of neutrophil lysates (inset, B). As a control, purified recombinant HBP was used in inset A.