Activated caspase-3 + IR in brain. Brain sections were stained with polyclonal cleave caspase-3 (Asp175) antibody, a marker of cell death. (A) Quantitation of caspase-3 + IR in cortex. The number of caspase-3 + IR cells in cortex was increased by ethanol, poly I:C and sequential ethanol-poly I:C. (B) Quantitation of caspase-3 + IR in hippocampal dentate gyrus. The number of caspase-3 + IR cells in dentate gyrus was increased by ethanol, poly I:C and sequential ethanol-poly I:C. The results are the means ± SEM of two independent experiments performed with seven mice per group. *P <0.05, **P <0.01, compared with vehicle control. ##
P <0.01, compared with poly I:C. (C and D) Representative images of caspase-3 + IR in cortex (C) and dentate gyrus (D) in vehicle control and ethanol-poly I:C groups. Scale bar, 200 μm. To determine if caspase-3 + IR was within neurons, brain sections were double-stained with NeuN (a neuronal marker). (E) Confocal microscopy images of cortex (upper panels) and dentate gyrus (lower panels) in ethanol-poly I:C group. Immunolabeling was visualized by using Alexa Fluor 488 and 555. Confocal microscopy indicates that caspase-3 + IR cells in green (left panels) are NeuN positive in red (middle panels), as shown in the merged images (right panels) with arrows indicating yellow co-labeling of caspase-3 and NeuN. Insets are higher magnification of the merged images. Scale bar, 30 μm; inset 5 μm.