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Figure 3: RSA59 can directly infect CNS resident microglia in the spinal cord explants culture in absence of any peripheral immune cells. Two hundred micron thick slice cultures were prepared from 4-week old C57BL/6 mice. 24 hrs after postexplantation, cultures were infected with 20,000 PFU/mL of RSA59 and incubated for 24 hrs, 48 hrs, and 72 hrs. Mock-infected cultures were also maintained in the same culture condition for the same period post explantation. 24, 48, and 72 hrs postinfection mock and RSA59-infected slices were stained with Iba1. Iba1 staining (red fluorescence) shows CNS resident microglia in mock-infected (a, e, i) and RSA59-infected slice culture (b, f, j). Merged images (c, d, g, h, k, l) show colocalization of EGFP in Iba1-positive cells of RSA59 infected spinal cord white matter. EGFP fluorescence (green) identifies virus-infected cells, anti-Iba1 (red fluorescence) detects microglia and DAPI (blue fluorescence) stains nucleus in the merged images.