Fluoro-Jade B

Histological examination of the ischemic brain was performed by NeuN and Fluoro Jade B as described previously by our lab (Q. G. Zhang et al., 2008 (link)). Briefly, after perfusion with 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB), the brains were post-fixed, cryoprotected with 30% sucrose until they sank and frozen sectioned (20 μm) in the coronal plane of the dorsal hippocampus (∼2.5-4.5 mm posterior from bregma). Every fifth section was collected and utilized for staining. Staining for NeuN and Fluoro Jade B was performed using a mouse anti-NeuN monoclonal antibody (1:500, Chemicon, MA, USA) and Fluoro Jade B (AG310, Chemicon) as described in detail previously by our laboratory (Q. G. Zhang et al., 2008 (link)). Images were captured on an LSM510 Meta confocal laser microscope (Carl Zeiss, Thornwood, NY) as described previously by our laboratory (C. Wakade et al., 2008 (link)). Cells that positively stained with NeuN and negatively stained with Fluoro Jade B were identified as “surviving neurons”; in contrast, double-stained yellow-colored cells represent CA1 neurons undergoing degeneration.
TUNEL staining was performed on the free-floating coronal sections using the In Situ Cell Death Detection Kit (Roche, Penzberg, Germany) following the manufacturer's instruction. Briefly, after washing with 0.1 % PBS-Triton-X100, the slides were permeabilized with 10 μg/ml proteinase K in 10 mM Tris/HCl (pH 7.4) for 15 min, and incubated with TUNEL reaction mixture including enzyme solution (TdT) and Tetramethylrhodamine (TMR)-labeled TUNEL-positive nucleotides in a humidified chamber for 1 h at 37 °C. Slides for negative control were incubated with the label solution without terminal transferase for TUNEL. Samples were analyzed with a LSM510 Meta confocal microscope. For quantitative analyses, the number of surviving neurons, and TUNEL-positive cells per 250 μm length of medial CA1 pyramidal cell layer was counted bilaterally in 4-5 sections per animal to provide a single value for each animal. A Mean ± SE was calculated from the data in each group (n = 6-8 animals) and statistical analysis performed as described below.

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.