OPN expression parallels the time course of macrophage infiltration into the infarct, a late event in the development of cerebral infarcts. This suggests that the upregulation of OPN is delayed until brain matrix remodeling is underway. While delayed expression of OPN in stroke has been reported, there are little data on its role in cerebral injury early during cerebral I-R. OPN can exist in two forms: secreted and intracellular. sOPN can engage various receptors on the cell surface, stimulating signal transduction pathways and cell adhesion. Certain of these receptors are upregulated following transient global cerebral I-R. Extensive post-translational modifications can modify the interaction of OPN with other proteins. OPN can be cleaved by thrombin, exposing a cryptic attachment motif that is capable of engaging additional integrins. iOPN, which lacks the signal sequence that targets the protein to secretory vesicles, possibly due to a down-stream alternative translational initiation signal, is expressed in Nutlin-3 548472-68-0 dendritic cells and macrophages of the immune system. In patients with advanced stages of Alzheimer’s disease, iOPN levels are increased in pyramidal neurons compared to normal human brain ; the authors suggested that iOPN may play a role in cell cycle progression, neuronal remyelination, and/or the formation of protein aggregates in Alzheimer’s Disease. The role of iOPN in the cellular response to stroke has not been studied. Here, we evaluated OPN expression early after cerebral I-R in three different areas of the brain. Next, we determined if the form of OPN we detected in cortical brain tissue was secreted or intracellular. Lastly, we investigated OPN expression in the presence of acetaminophen, a drug recently shown to reduce apoptosis and mitochondrial dysfunction in early cerebral I-R. For immunocytochemical analysis, sections were first washed in PBS. Endogenous hydrogen peroxidases were quenched using 0.3% H2O2 in PBS for 30 min. Sections were then blocked in 1% BSA for 1 h followed by incubation with 2A1 overnight at 4uC in a humidified chamber. The 2A1 antibody used in this study was generated in our laboratory; it binds to an epitope centrally located in the C-terminal thrombin cleavage fragment about 50 amino acids downstream of the RGD integrin-binding site. The epitope is not subject to post-translational modifications that can interfere with antibody-epitope recognition. A few sections were used as negative controls, where the primary antibody was omitted. Slides were then incubated with secondary HRPconjugated goat anti-mouse antibody for 1 h. Immunostaining was developed by the peroxidaseantiperoxidase procedure using 3-39-diaminobenzidine as the cosubstrate. Sections were counterstained with hematoxylin before mounting.