Histochem Cell Biol (2008) 130:281–297 285 Fig. 3 Cytochemical detection of NBT-derived diformazan reaction product by reﬂected-light laser scanning confocal microscopy. Two neutrophils that were incubated with NBT in the absence of exogenous stimulating agents are shown. a Cells were viewed by non-confocal transmitted light. b The same cells were imaged by laser scanning confocal microscopy in the reﬂectance mode. The adherent neutrophils have a region of cell–cell contact. The reaction product, indicative of O2- production, is detected in both the transmitted light image and the confocal reﬂectance image (arrows). c The co- phagosome (Klebanoff 1970). The activity of MPO in the phagosome of intact neutrophils was detected with 3,30- diaminobenzidine (DAB). Deposits of reduced DAB in the phagosome strongly suggested that endogenous H2O2 was produced within the phagosome since exogenous H2O2 was not added to the cytochemical medium (Briggs et al. 1975a). Other DAB-based cytochemical methods for the localization of ROS have been developed (see below). The presence of MPO in the phagosome is important for the production of the full complement of microbicidal agents. The demonstration of MPO delivery to neutrophil phagosomes at the electron microscope level is shown (Fig. 4). Detection of hydrogen peroxide with cerium Hydrogen peroxide, produced by the rapid dismutation of superoxide, was initially detected at the ultrastructural localization of the transmitted light image and the confocal reﬂec- tance image is the pseudocolor merge of the two images; the reaction product is indicated by the arrow. d A color wedge has been inserted to estimate the intensity of the reﬂectance signal. Note that the diformazan reaction product is almost entirely restricted to the area of cell–cell contact, indicating the lack of a global signal transduction cascade leading to the generation of O2- over the entirety of the cells. Bar: 10 lm. Reprinted from Robinson and Badwey 1995 level using a cerium-based cytochemical reaction (Briggs et al. 1975b). The cerium-based reaction proceeds as follows: NADPH þ 2O2 ! NADPþ þ Hþ þ 2O2 2O2 þ 2Hþ ! H2O2 þ O2 H2O2 þ CeCl3 þ 3OH ! CeðOHÞ2OOH þ H2O þ 3Cl In this reaction, sequence endogenous phagocyte NADPH is utilized by the NADPH oxidase to generate O2-. The O2- subsequently undergoes dismutation to form H2O2 which, in turn, reacts with cerium to form cerium perhydroxide, an electron dense product detectable in the electron micro- scope. Cerium perhydoxide is a ﬁne, non-crystalline, electron-dense reaction product that generally forms with low background. Thus the cerium reaction is ideal for electron microscopy. However, the cerium perhydroxide reaction product was not initially considered useful for 123