In each region, six random images from each brain sample were captured within a standard ROI, the density of immunostaining and fluorescence was measured in pixels within mostly this area. Subsequently, the average of the six measurements was used to represent the immunoreac tivity or fluorescence intensity of each sample. When measuring fluorescence intensity in the cells, we elimi nated the background by adjusting threshold to avoid background staining. For IR cells counting, a modified stereological method was used to quantify cells within regions of interest following immunostaining of brain sections using the CAST stereological system.

Specifically, cell density of caspase 3 and gp91phox immunoreactivity was determined following the optical disector method, which was calculated as follows, Where ��Q is the sum of Inhibitors,Modulators,Libraries the caspase 3 or gp91phox IR cells counted from each disector frame, ��disector is the Inhibitors,Modulators,Libraries sum of the number of disector frames counted, A is the known area associated with each disector Inhibitors,Modulators,Libraries frame, and h is the known distance between two disec tor planes. For colabeling study, double or triple stained sections were digitally photographed with Leica SP2 AOBS con focal microscope and analyzed with Leica SP2 LCS software. Statistical analysis The data are expressed as mean SEM and statistical significance was assessed with an ANOVA followed by Bonferronis t test using the StatView program. A value of P 0. 05 was con sidered statistically significant.

Results Chronic ethanol increases caspase 3 expression and Fluoro Jade B staining To determine the effect of ethanol exposure on neuro degeneration in mice, immunohistochemistry for cleaved Inhibitors,Modulators,Libraries caspase 3 and Fluoro Jade B histochemistry were performed on C57BL 6 mouse brain sections treated with water or ethanol daily for 10 days. Ethanol treated Inhibitors,Modulators,Libraries mice showed an increase in activated caspase 3 immunoreactivity 24 hours after the last dose of ethanol treatment, compared to water controls. The number of activated caspase 3 immunor eactive cells increased 3. 1 fold in cortex and 3. 5 fold in dentate gyrus in ethanol treated mice. To determine if caspase 3 immunoreactivity was neuronal, double immunohistochemistry for cleaved caspase 3 and Neu N, a neuronal marker was used. Confocal microscopy indicated that most activated cas pase 3 IR cells colocalize with Neu N IR cells, suggesting chronic ethanol exposure causes neuro nal cell death.

Fluoro Jade B, another cell death marker, was also used to assess ethanol induced neurotoxicity. Brain sections from control animals showed little Multiple myeloma or no Fluoro Jade B staining. However, mouse brains exposed to chronic ethanol increased 10 fold in cortex and 7. 6 fold in dentate gyrus in intensity of Fluoro Jade B positive staining 24 hours after the last dose of etha nol treatment compared to water control group. Confocal microscopy indicated that most Fluoro Jade B positive cells were colocalized with Neu N IR.