Histochem Cell Biol (2008) 130:281–297 localization of cytochrome b in neutrophils and macro- phages has been carried out using immunoﬂuorescence microscopy. Resting neutrophils showed a primarily intracellular granular labeling pattern while macrophages did not display an intracellular labeling pattern but instead showed a cell surface pattern (Johansson et al. 1995). While not a component of the NADPH oxidase, myelo- peroxidase is important in the formation of downstream ROS and for participating in microbicidal activity of neu- trophils. Fluorescence microscopy and ﬂow cytochemistry have been used to analyze binding of quantum dots con- jugated with anti-myeloperoxidase (Hoshino et al. 2007). The Hoshino study reports that these quantum dots bind neutrophils stimulated with proinﬂammatory cytokines but not control cells. Immunolocalization of components of the NADPH has also been carried out at the electron microscope level. Neutrophils that were cryoﬁxed and prepared by mole- cular distillation drying were used in conjunction with antibodies to components of the cytochrome b558; it was found that *20% of the immunolabel was associated with the plasma membrane with the remainder associated with cytoplasmic granules (Jesaitis et al. 1990). When ultrathin cryosections were used as the labeling substrate for a monoclonal antibody to the 22kD component of cytochrome b558, there was little labeling associated with the plasma membrane with the label being primarily associated with the speciﬁc granules (Ginsel et al. 1990). In a subsequent immunoelectron microscopy study, cytochrome b558 co-localized to a signiﬁcant extent with albumin (Calafat et al. 1993). Albumin was previously shown to be a marker for the matrix of the secretory vesicle of neutrophils (Borregaard et al. 1992). This secretory vesicle is the same structure as the alkaline phosphatase-positive vesicle we described using enzyme cytochemistry (Kobayashi and Robinson 1991; Robinson et al. 1999). Cytochrome b558 has been reported as a component of the speciﬁc granule, the gelatinase granule, and secretory vesicles of the human neutrophil (reviewed in Borregaard and Cowland 1997). The bacterium Ana- plasma phagocytophilum invades neutrophils but appears to repress the respiratory burst. Immunoelectron micro- scopy showed that both subunits of cytochrome b558 were signiﬁcantly reduced in phagosomes containing A. phagocytophilum when compared to E. coli containing phagosomes and thus could be part of the explanation for the reduced respiratory burst (IJdo and Mueller 2004). Combined immunoelectron and immunoﬂuorescence microscopy of neutrophils infected with A. phagocyto- philum also demonstrated a low association of cytochrome b558 subunits with phagosomes containing A. phagocytophilum (Carlyon et al. 2004). These two studies with A. phagocytophilum illustrate the importance 289 of immunolocalization in understanding the interaction of particular microbes and the neutrophil phagosome. Immunolocalization experiments and particularly dou- ble or triple localization preparations can be very valuable since low afﬁnity interactions that may be detected by immunolocalization techniques may not survive the harsher conditions associated with cell-fractionation stud- ies. For example, as noted above, transient recruitment of p47phox and p67phox to phagosomes in neutrophils from X-linked CGD was reported (Allen et al. 1999); cell frac- tionation and cell-free activation systems from these cells did not show translocation of these components (Heyworth et al. 1991). Fluorescence-based localization of ROS The detection of ROS by ﬂuorescence microscopy is an attractive option since it permits evaluation of single-living phagocytes. Endogenous autoﬂuorescence associated with NADPH and NADH can be monitored and a change in the distribution of NAD(P)H at phagosomes has been observed (Liang and Petty 1992). It is noteworthy that polarized substratum-attached neutrophils produce bursts of O2- production (determined using an exogenous ﬂuorescent probe for ROS) that correlated with waves of NAD(P)H [determined by monitoring the autoﬂuorescence of NAD(P)H] that traveled from the rear to the front of crawling cells (Kindzelskii and Petty 2002). In this case, cells were attached to quartz microscope slides. Production of O2- occurred in control cells as well as in cells stimu- lated with a chemotactic peptide. The difference between the NAD(P)H wave and the release of O2- in the two conditions was the periodicity; it was *22 s in the absence of the chemotactic peptide and *11 s in its presence (Kindzelskii and Petty 2002). It has long been known that in standard biochemical assays for monitoring O2- pro- duction in populations of cells in suspension, that these cells are quiescent (see Fig. 1). How are these two obser- vations to be reconciled? In the case of crawling neutrophils, they have undergone a dramatic shape change. On the other hand, neutrophils assayed in suspension have essentially the same rounded shape that they have in the bloodstream. However, when intact neutrophils, in sus- pension, are challenged with a stimulus (e.g., phagocytizable particles, phorbol myristate acetate, the chemotactic peptide f-Met-Leu-Phe, arachidonic acid, or retinoids) they too undergo changes in shape and reorga- nization of intracellular compartments (e.g., Badwey et al. 1984; Robinson et al. 1987; Kobayashi and Robinson 1991). The degree to which shape change itself modulates the phagocyte NADPH oxidase is not clear. Nathan (1987) has shown that tumor necrosis factors did not induce pro- duction of H2O2 in neutrophils in suspension but when 123