Assessment of negative control signals in immunohistochemical experiments. To ensure that visualization of astrocytes and microglia was not confounded by nonspecific secondary antibody binding, a negative control for the Cy2 (donkey-anti-mouse DyLight 488) and Cy5 (donkey-anti-rabbit DyLight 649) channels was included with primary antibodies omitted. Individual channels are presented as inverted gray scale. Weak, diffuse background signal was observed in the Cy2 channel, and no signal was apparent in the Cy5 channel. No nonspecific cell staining in the absence of primary antibodies occurred. Blue: Hoechst; Green: Cy2; Red: Cy5.