Figure 5

Immunofluorescence and confocal microscopical analysis of p47^phoxlocalization in LPS-stimulated microlgia cells. HAPI cells were treated with LPS for 10 min in the absence or presence of SN pretreatment for 0.5 h. Cells were immunostained with a rabbit polyclonal antibody against p47^phox, then washed and incubated with FITC-conjugated goat anti-rabbit antibody. The signal of p47^phox(FITC-p47^phox; on left) and the merge view of cell morphology and p47^phox(Phase plus FITC-p47^phox, on right) are shown. The inset shown in the right corner of each treatment condition shows the location of FITC-p47^phoxin a single, randomly selected cell. Focal planes spaced at 0.4-μm intervals were imaged with a Zeiss 510 laser scanning confocal microscope (63 × PlanApo 1.4 numerical aperture objective) equipped with LSM510 digital imaging software. Three adjacent focal planes were averaged using Metamorph software. The signal of p47^phox(FITC-p47^phox; green) and the merge view of cell morphology and p47^phox(Phase plus FITC-p47^phox) are shown. Scale bar, 50 μm.