Immunohistochemical localization of the class III histone deacetylase SIRT1 (Sir2) in the murine enteric nervous system. A. Confocal image of a whole mount preparation of colon stained with a goat antibody to the neuronal marker human neuronal protein (HuD; 1:100; Santa Cruz; sc-5977; green). HuD immunoreactivity is displayed by neurons in a myenteric ganglion. B. Double label confocal image of the same area depicted in A stained with an antibody to HuD and a SIRT1-specific antibody made in rabbit (1:500; Abcam Inc. Cambridge MA; ab 16640). Myenteric neurons display both HuD (green) and nuclear SIRT1 immunoreactivity (red). For whole-mount preparations, segments of colon were cut along the mesenteric border and the resulting sheet of gut was pinned flat, mucosal side up, in a silicone elastomer (Sylgard, Dow Corning, Midland, MI)-coated dish. The tissue was fixed for 3 hours with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After fixation, the preparations were washed in phosphate-buffered saline (PBS) for 1 hour and whole-mount preparations of longitudinal muscle with adherent myenteric plexus (LMMP) were generated as previously described . Non-specific binding was blocked by incubating the preparations with 6% (v/v) normal horse serum, with Triton X-100 (0.5%), in PBS for 60 minutes. The preparations were then exposed for 24 h to primary antibodies at 4°C. After washing with PBS, sites of bound primary antibodies were detected by incubation with donkey anti-rabbit or donkey anti-goat secondary antibodies coupled to DyLight™ 549 (1:400; Jackson ImmunoResearch Labs.West Grove, PA) or DyLight™ 488 (1:400; Jackson ImmunoResearch Labs.) for 3 hours. Confocal images were obtained using an Olympus FluoView FV300 confocal microscope. Scale bar, 30 μm.