KCNN /SK expression in cultured microglia. A. Relative mRNA expression of KCNN1-KCNN3 was determined by quantitative real-time RT-PCR (qRT-PCR), and normalized to the housekeeping gene, TATA box-binding protein (TBP). Bars represent mean ± SEM for 3 cultures from separate rat litters. After microglia were activated with lipopolysaccharide (LPS; 100 ng/ml, 24 h), KCNN3 expression increased (†p < 0.5) and was higher than KCNN1 and KCNN2 expression (**p < 0.01; ***p < 0.001). The inset shows a typical, essentially pure microglial culture labeled with FITC-conjugated tomato lectin. B. SK3 immunostaining in unstimulated microglia. Cultured microglia were labeled with anti-SK3 (rabbit polyclonal, 1:200), a Cy3-conjugated secondary (donkey anti-rabbit, 1:400; red), the microglia/macrophage stain, FITC-conjugated tomato lectin (1:500; green) and the nuclear marker, DAPI (1:3000; blue). Scale bars, 10 μm. C. Specificity of the SK3 staining. Upper panels: Lack of non-specific staining (no SK3 primary antibody) in microglia, which were labeled with DAPI (blue), tomato lectin (green) and the Cy3-conjugated secondary antibody (left). The color-separated image at the right shows the absence of non-specific staining in the Cy3 (red) channel. Scale bar, 10 μm. Lower panels: Phase-contrast images show SK3 staining in transfected (left), but not in non-transfected CHO cells (anti-SK3 and Cy3-conjugated secondary antibodies, as in panel B). Scale bars, 30 μm.