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.
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.
