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Double staining protocols using the most popular immunoperoxidase techniques may raise difficulties. The two ordinary detection systems may cross-talk, when the primary antibodies are derived from phylogenetically closely related animals. A color shift of the 3,3′-diaminobenzidine (DAB) polymer may occur during the second development, resulting in poor distinction between the two kinds of deposits. A post-DAB technique, sulfide-silver-gold intensification, was fine tuned to eliminate these difficulties, which may be especially suitable for colocalization of cell nuclei and perikarya of the same cells. The revised method was probed in combination with a subsequent other immunoperoxidase step or fluorochrome-tagged reagents. The nuclear antigens (BrdU, c-Fos, and Prox-1) were first visualized with DAB polymer, which were then treated with SSGI, turning the deposit black. Thereafter, cytoplasmic antigens (doublecortin, neuronal nuclei, and calbindin) were detected with either another immunoperoxidase using DAB again or immunofluorescence labeling. In both approaches, the immunopositive nuclei and cytoplasmic sites could be easily distinguished even at low magnifications. Different shielding or eluting posttreatments were compared for consecutive acetylcholinesterase histochemistry terminated with DAB development and immunohistochemistry in the same sections. In conclusion, we recommend post-DAB treatments that abolish interactions between detection systems and allow clear distinction between the two signals under various conditions

Defensins are an important family of natural antimicrobial peptides. Chlamydophila pneumoniae, a common cause of acute respiratory infection, has a tendency to cause persistent inflammatory diseases such as atherosclerosis, which may lead to cardiovascular disease or stroke. As endothelial cells are related to the physiopathology of stroke, the effects of in vitro C. pneumoniae infection on the expression of human β-defensin 2 (HBD-2) in brain capillary endothelial cells (BB19) was investigated. A time-dependent increase in HBD-2 mRNA was observed by means of real-time reverse transcription PCR (RT-PCR) in BB19 cells following C. pneumoniae infection, with a maximum increase at 24 h. A gradual induction of HBD-2 protein in the C. pneumoniae-infected endothelial cells was detected by immunoblotting. Immunofluorescence revealed the staining of HBD-2 in the cytoplasm of endothelial cells following C. pneumoniae infection. The secretion of HBD-2 (confirmed by ELISA) was significantly elevated 24 h after C. pneumoniae infection. These novel results indicate that HBD-2 is expressed and produced in the human brain capillary endothelial cells upon infection with C. pneumoniae, and provide evidence that HBD-2 plays a role in the early immune responses to C. pneumoniae and probably in the immunopathogenesis of atherosclerosis.