Eastern Equine Encephalitis in Children, Massachusetts and New Hampshire,USA, 1970–2010
Michael A. Silverman, John Misasi, Sandra Smole, Henry A. Feldman, Adam B. Cohen, Sandro Santagata, Michael McManus, and Asim A. Ahmed
Author affiliations: Author affiliations: Children's Hospital Boston, Boston, Massachusetts, USA (M.A. Silverman, J. Misasi, H.A. Feldman, S. Santagata, M. McManus, A.A. Ahmed); Department of Public Health, Boston (S. Smole); Massachusetts General Hospital, Boston (A.B. Cohen); Brigham and Women’s Hospital, Boston (S. Santagata)
Figure 4. . . . . . Eastern equine encephalitis virus (EEEV) colocalized with neurons in patient 12 in a study of children with eastern equine encephalitis (EEE), Massachusetts and New Hampshire, 1970–2010. A) Immunohistochemistry, using EEEV immune ascites, of the entorhinal temporal cortex, demonstrating EEEV-infected neurons (arrow) (magnification ×400). B) Dual immunohistochemistry with EEE immune ascites (red stain) and a mouse monoclonal anti–neuronal nuclei (NeuN) antibody (brown stain) demonstrates that EEE-infected cells are NeuN–expressing neurons (arrows) (magnification ×400). C) Dual immunohistochemistry with EEE immune ascites (red stain) and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (brown stain) demonstrated that EEE-infected cells (arrow) do not express GFAP and are therefore not glial cells (arrowhead) (magnification ×400). Single antibody immunohistochemistry was performed with heat-induced antigen retrieval, using pressure cooker treatment (120°C for 30 s at 15 psi) with citrate buffer, pH 6.0. Lyophilized ascites from American Type Culture Collection (Manassas, VA, USA) was resuspended in 1 mL of double-distilled H20 and stored as stock solutions at −20°C. The ascites stocks were applied at 1:500 for 40 min at room temperature, after which labeled horseradish peroxidase (HRP) anti-mouse antibody was added, and the stocks were incubated 30 min at room temperature. Visualization was performed by using 3,3′-diaminobenzidine chromogen (Dako EnVision+ System-HRP (DAB); Dakocytomation, Carpinteria, CA, USA). Dual immunohistochemistry was performed by using polyclonal rabbit antibody to GFAP (DAKO, Carpinteria CA, USA) at 1:20,000 and mouse monoclonal anti-NeuN, clone A60, MAB377 (Millipore, Billerica, MA, USA) at 1:7,500. Both antibodies were visualized by using the Dako EnVision+ System-HRP (DAB)-brown, and then EEE immune ascites (1:500) was applied, using the alkaline phosphatase method, and visualized again by using Permanent Red (Dako).
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