FluoroJade B staining of degenerating cells was conducted following published methods (Morris et al., 2009 ; Schmued and Hopkins, 2000 (link)). Briefly, every 12th section was mounted to Superfrost Plus® slides (Fisher Scientific, Waltham, MA) and dried overnight. Slides were processed through graded alcohols, rinsed in dH20 then incubated in 0.06% KMnO4 for 10 minutes. Following a wash in dH20, sections were then incubated in FJB (Chemicon, Temecula CA) in the dark. After additional washes in dH20, sections were dried then coverslipped in Cytoseal® (Stephens Scientific, Wayne, NJ). Quantification of FJB-positive (FJB+) cells was conducted on an Olympus BX-51 microscope fitted with epifluorescence including a 488λ cube for blue light excitation. A profile counting methodology was used to quantify all cells visualized within the region of interest. This method was chosen over unbiased stereological techniques because of difficulty in accurately detecting section thickness in sections with very low background staining as our FJB method produces and to compare across regions where stereology is not appropriate. Further, we have shown previously that profile methodologies result in identical percent change between control and experimental groups (Crews et al., 2004 (link)), and relative difference is the question of interest in this study. FJB+ cells were counted in brain regions that consistently show alcohol-induced cell death in this model: the hippocampal dentate gyrus (granular cell layer and subgranular zone combined), the entorhinal and perirhinal cortices combined and the piriform cortex (Obernier et al., 2002a (link); Obernier et al., 2002b (link)). Specifically, in the dentate gyrus, each blade was counted and averaged separately and the mean number of FJB+ cells per dentate gyrus section was calculated by adding the averages from the superior and inferior blades together. FJB+ cells are reported as mean number of cells per section ± SEM.