Murine primary cortical mixed glial culture were treated with LPS (1 μg/ml) and IFNγ (0.5 ng/ml) for 24 hours and immunostained for NOS2 (A, B), GFAP (C) or CD11b (D). A and C show the same field and E is their merged image. B and D show the same field and F is their merged image. In control cultures NOS2-immunoreactive cells were not observed (data not shown). There is (LPS + IFNγ)-induced NOS2 expression in numerous cells (A, B). NOS2-positive cells were almost never GFAP immunoreactive (A, C, E) indicating a lack of NOS2 expression in most astrocytes. In contrast, virtually all NOS2-positive cells (>98%) were identified as microglia by their CD11b immunoreactivity. Nuclei are counterstained with Hoechst 33258 in A-D. NOS2-positive cells were identified with a rabbit anti-NOS2 antibody (1:500, BD Biosciences), GFAP-positive cells with a mouse anti-GFAP antibody (1:1000, Sigma) and CD11b-positive cells with 5C6 mouse anti-CD11b antibody (1:400, Serotec). Bar, 100 μm.