Histochem Cell Biol (2008) 130:281–297 cell is the norm. The asymmetric delivery of ROS was also noted in neutrophils attached to aggregated IgG associated with glomerular basement membranes; in this case the reaction product, indicative of H2O2 production, was on the side of the neutrophil attached to the non-phagocytizable basement membrane (Vissers et al. 1985). The asymmetric generation of ROS has been observed by ﬂuorescence microscopy (see more below) in polarized neutrophils (i.e., crawling over a substratum) (Kindzelskii and Petty 2002). We also observed polarized distribution of cerium perhy- droxide, indicative of H2O2 production, in crawling cells (Heyworth et al. 1997). This appears be similar to the sit- uation reported by Kindzelskii and Petty (2002) who used ﬂuorescence microscopy. Another example of the asym- metric generation of ROS was previously shown herein using NBT as the detection reagent; in this case, O2- was detected where two polarized cells made contact (see Fig. 3). These ﬁndings, enabled by microscopy, indicate the complexity of the regulation of production of ROS by phagocytic leukocytes and raise a number of intriguing questions related to the cell biology and biochemistry of the respiratory burst. Such asymmetric and/or polarized generation of ROS may have important implications in pathophysiology. It is generally considered that the activated NADPH oxidase is associated with the plasma membrane or its derivative the phagosome (e.g., Nauseef 2004). However, there are several ﬁndings that suggest that there may be a separate intracellular pool of NADPH oxidase activity (Lundqvist et al. 1996; Kobayashi et al. 1998; Lundqvist- Gustafsson and Bengtsson 1999; Vaissiere et al. 1999; Karlsson et al. 2000; Karlsson and Dahlgren 2002; Brown et al. 2003; Ambruso et al. 2004). There are often small vesicle-like structures containing cerium perhydroxide indicative of the production of H2O2 in cells that have phagocytized particles (Fig. 6). These structures can vary in number from one thin section to another in conven- tional electron microscopy such that some sections have numerous cerium perhydroxide-containing vesicles and other sections have few or even none of the positive vesicles. A detailed analysis of serially sectioned neutro- phils that had internalized particles and in which the localization of H2O2 had also been carried out, would be required to address this question adequately and to understand further this intracellular pool of NADPH oxidase in neutrophils following phagocytosis. Insofar as the author knows, such an analysis has not been con- ducted. The exact nature of these vesicles containing reaction product needs further investigation to determine if they are intracellular in origin or whether they are derived from the plasma membrane since it has been reported that a burst of pinocytosis accompanies phago- cytosis (Botelho et al. 2002). However, even if these 287 Fig. 6 Electron micrographs of portions of two different neutrophils that ingested zymosan particles; sites of H2O2 generation were detected with the cerium-based cytochemical reaction. a A portion of a cell is shown in which phagosomes show a spectrum of responses: P1, heavy reaction product; P2, moderate reaction product; P3, little reaction product. There are several small vesicular structures containing reaction product in the periphagosome region (arrows) suggesting intracellular sites of H2O2. Patches of reaction product are at the cell surface (arrowhead). Portions of the nucleus (N) are visible. b A portion of a cell is shown in which phagosomes show different responses: P1, heavy reaction product; P2 and P3, little reaction product. There are several small vesicular structures containing reaction product in the periphagosome region (arrows) suggesting intracellular sites of H2O2. Patches of reaction product are at the cell surface (arrowhead). Portions of the nucleus (N) are visible. Bars: 1 lm 123