Fig. 6.
Confocal imaging of 5 integrin in control and [Ca++]i-buffered PMNs plated on fibronectin.
PMNs were either loaded with quin2/AM (E-H) or not (A-D), then prepared for immunofluorescence as in Figure 3. Samples were incubated with 10 μg/mL of a monoclonal antibody to the extracellular domain of α5 integrin, rinsed with PBS, and then incubated with a TRITC-conjugated secondary antibody. The samples were viewed using a Bio-Rad MRC 600 laser scanning confocal microscope and vertical sections were obtained. x-y (A, E) and x-z (B, F) projections of both control (A-D) and quin2-buffered (E-H) cells are shown. Panels C and G show the localization of α5 at a single x-y slice at the depth indicated by the arrows in panels B and F, respectively. Panels D and H show a single x-z slice along the axis of the cells indicated by the arrows in panels A and E, respectively. Bar = 10 μm.