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1% Triton X-100. Slides were incubated with primary antibodies (Table?2) in blocking solution overnight at 4��C, washed to remove unbound antibody, and incubated with species-appropriate fluorescence-conjugated secondary antibodies (Table?2) in blocking buffer. To control for non-specific secondary antibody signals and background http://www.selleckchem.com/products/AZD0530.html fluorescence, a negative control was included with primary antibodies omitted. Nuclei were stained with Hoechst 33258 (5?��g/mL; Invitrogen). After washing, slides were coverslipped with Aqua Poly/Mount (Polysciences, Warrington, PA, USA) and dried prior to imaging. Images were acquired using a confocal laser scanning microscope (Leica TCS SP2 AOBS, Exton, PA, USA) equipped with UV-diode (Hoechst, 405?nm), argon (488?nm), and helium-neon (546 and 649?nm) lasers. To characterize MAI expression within hippocampal subregions, tissue sections were imaged as 1024?��?1024 pixel, 3.9?��m series of six optical sections http://www.selleckchem.com/products/GDC-0941.html (step size?=?0.65?��m) acquired using a 20�� oil-immersion objective with 2?��?2 line and frame averaging to decrease noise, using identical laser settings optimized for each antibody. High-resolution imaging of Hoechst nuclear staining and neurofilament/MAI immunoreactivity was performed using a 63�� oil-immersion objective with 2?��?2 line and frame averaging, with identical laser settings optimized for each antibody. All images are presented as average projections of z-stacks. Additional noise reduction and background subtraction were performed using Adobe Photoshop CS4 software (Adobe Systems, San Jose, CA, USA), with equal adjustments applied to all images of a given antibody. 20�� images of MAI distribution within http://en.wikipedia.org/wiki/Diglyceride hippocampal subregions were converted to inverted grayscale to enhance contrast in monochrome images. Hippocampus-dependent cognitive performance of adult and aged Fischer 344?��?Brown Norway rats was assessed by Morris water maze testing, and aged animals were assigned to cognitively intact or cognitively impaired groups based on mean proximity to the escape platform during probe trials as previously described (Freeman et?al. 2009; VanGuilder et?al. 2011b). Stratification of aged rats into cognitively intact and cognitively impaired groups was evident in the three cohorts included in this study (Table?1). Statistical analysis confirmed that spatial learning and memory was superior in adult and aged cognitively intact rats compared with aged cognitively impaired rats (p?