Figure 9
Determination of cell phenotype for NOX subunit gp91 phox expression and O 2 - production. Cell phenotype was determined by confocal microscopy of sections triple labeled for gp91phox (blue), O2 - (red) with Iba-1, or Neu-N or GFAP (green). C57BL/6 mice were injected with dehydroethidium (10 mg/kg, i.p.) 23.5 hrs after ethanol treatment. Brains were harvested 30 min later and frozen sections (15 μm) were processed and double-immunostained for gp91phox and Iba1, gp91phox and Neu-N as well as gp91phox and GFAP to analyze triple labeling or colabeling by using the Leica SP2 LCS confocal software. Confocal microscopy shows that gp91phox +IR cells are triple-labeled with O2 - and Iba1 or Neu-N, but not with GFAP. Triple-labeled representative images are shown in white with arrows indicating the triple-labeling of gp91phox with O2 - and Iba1 (merged, the lower left panel) or gp91phox with O2 - and Neu-N (merged, the lower middle panel). Double-labeled representative images are shown in pink with arrowheads indicating the colabeling of gp91phox with O2 - , but not with GFAP (merged, the lower right panel). The images presented are from dentate gyrus of ethanol treated mice. Scale bar = 20 μm.