288 vesicles containing reaction product showing H2O2 pro- duction are pinocytotic in origin, there remains the question of how (and when) are the cytosolic components of the oxidase directed to these sites. O2- detection with DAB-Mn2+ A cytochemical method for the ultrastructural localization of O2- was developed; the cytochemical medium contains DAB-Mn2+ as the key components (Briggs et al. 1986). Subsequent study showed that this method was speciﬁc for O2- and was not responsive to 1O2 (Steinbeck et al. 1993). A modiﬁcation of this methodology was used to localize sites of superoxide production in rat endothelium in situ (Babbs et al. 1991a, b). The DAB-Mn2+ procedure along with modiﬁcation of this DAB method has been reviewed (Babbs 1994). The DAB-Mn2+ method was used to ana- lyze intracellular sites of O2- in phorbol ester-activated neutrophils (Kobayashi et al. 1998). This latter study generated interest since it identiﬁed intracellular sites of O2- production as being distinct from plasma membrane derived vesicles. Two alternative explanations could be posited for this observation. One explanation is that bona ﬁde sites of NADPH oxidase assembly and activation occur on certain intracellular compartments; the second expla- nation is that the cytochemical reaction occurs in vesicles that were internalized and are in reality derived from the plasma membrane and do not represent an intracellular pool of NADPH oxidase. To address the source of the O2- - positive compartments, Kobayashi and colleagues used native and cationic ferritin in conjunction with O2- locali- zation. Ferritin was used to mark plasma membrane- derived endocytic vesicles. Sites of O2- production were distinct from ferritin-positive vesicles, indicating that these sites of O2- production were not derived from the plasma membrane. However, the O2- - positive compartments and the ferritin-positive compartments did fuse and their con- tents mix at later times. This illustrates the complex patterns of intracellular membrane trafﬁcking in activated neutrophils. The Kobayashi study also combined O2- cytochemistry and cerium-based cytochemistry for the detection of alkaline phosphatase. This was possible since the electron-dense reaction products generated with these two cytochemical reactions were distinct in their mor- phology. It was found that these two cytochemical reactions occurred in the same compartment. Moreover, the kinetics of phorbol ester-induced O2- generation (as mea- sured biochemically) and the up regulation of alkaline phosphatase activity to the cell surface (as measured bio- chemically) occurred with a similar time course. This further supports the contention that intracellular generation of O2- is associated with the alkaline phosphatase-positive compartment. Previous work had shown that alkaline 123 Histochem Cell Biol (2008) 130:281–297 phosphatase was unregulated to the cell surface in response to chemoattractants (Borregaard et al. 1987). Subsequently it was shown that alkaline phosphatase resides in unique rod-shaped structures that are in lower abundance than azurophil or speciﬁc granules and in response to chemo- attractants these structures appeared to fuse into longer tubular compartments that also fused with the plasma membrane (Kobayashi and Robinson 1991; Robinson et al. 1999). Immunocytochemical localization of components of the NADPH oxidase Immunocytochemical localization of NADPH oxidase components has been important in understanding the assembly of the NADPH oxidase. The rates of ROS gene- ration by neutrophils during phagocytosis, with two different types of particles, were measured biochemically and compared to the kinetics of the association of cyto- chrome b558 and p47phox and p67phox with phagosomes as determined by immunoﬂuorescence microscopy (DeLeo et al. 1999). It was found that cytochromes b558 and p47phox and p67phox are associated with phagosomes and that p47phox and p67phox subsequently dissociated from the phagosome; this later event correlated with the end of the oxidase activity. This same group, using immunoﬂuores- cence microscopy, showed in neutrophils from patients with X-linked CGD that p47phox and p67phox accumulated in the periphagosomal region but did not remain when phagocytosis was completed (Allen et al. 1999). In con- trast, phagocytosis in normal neutrophils was accompanied by a more stable assembly of p47phox and p67phox at the phagosome membrane. These data suggested that cyto- chrome b558, non-functional in X-linked CGD, was required for more stable association of these cytosolic components with the phagocyte membrane. Immunoﬂuo- rescence microscopy was used to show that gp91phox and cytosolic phospholipase A co-localize to the plasma membrane in stimulated PLB-985 cells (a neutrophil sur- rogate) and that in the absence of gp91phox phospholipase A does not translocate to the plasma membrane (Shmelzer et al. 2003). The movement of gp91phox in PMA stimulated neutrophils has been described with a set of monoclonal antibodies using immunoﬂuorescence microscopy; one of these antibodies appeared to bind preferentially to the active form of cytochrome b558 (Campion et al. 2007). Biochemical and immunoﬂuorescence microscopy experi- ments were used to study the interactions of p47phox and interleukin-1 receptor-associated kinase-4 (IRAK-4) in neutrophils treated with lipopolysaccharide (LPS) (Pacqu- elet et al. 2007). The authors report co-localization of p47phox and IRAK-4 at sites near the plasma membrane in LPS stimulated neutrophils. Comparison of the sub-cellular