Fig 1: (a–c) Active lesion in a patient with primary progressive multiple sclerosis. (a) Luxol fast blue (LFB) myelin staining shows a demyelinated lesion with defined borders. (b) In the adjacent section stained for p22phox intense expression is seen at the active lesion edge, spanning into the adjacent normal appearing white matter (initial lesion area). (c) In the inactive centre of the lesion p22phox is weakly expressed in some macrophages. [d–h(hh)] These images show the expression of oxidative burst associated molecules in the normal appearing white matter (left column), in the zone of initial tissue injury (centre column) and in the demyelinated zone (right column). Most pronounced expression of all proteins is seen in the initial lesion area (centre panel), while expression for p22phox and gp91phox is much weaker in lipid containing macrophages in the lesion centre (right panel). In the normal appearing white matter microglia nodules can be seen, which are intensely stained for p22phox, gp91phox and NOX1. P22phox, gp91phox and p47phox are only expressed in macrophages and microglia (see below), while Nox1 shows a broader expression also in astrocytes (asterisk in gg) and endothelial cells (asterisk labels the vessel with endothelial staining); the expression of Noxo1 is even broader compared with that of Nox1. P47phox staining is absent in the normal appearing white matter (g), while intense expression is seen in macrophages and small microglia like cells; the insert shows expression of p47phox (red) in macrophages stained with LN3 (green). In the lesion centre, only weak reactivity for p47phox is seen, mainly in perivascular macrophages (at higher magnification in the inset). (l–o) Confocal laser microscope images of double staining with different Nox markers and CNS cell-specific markers; the staining combinations are indicated on the figures. These data show co-localization of different Nox components within the same macrophages or microglia cells in the multiple sclerosis lesions. Nox 1 is also expressed in GFAP-positive astrocytes (n) in the absence of p22phox (o). Red and green staining depicts the individual antigens as indicated in the figure; yellow staining represents double staining.