Immunohistochemistry and Confocal Imaging Method for immunohistochemical procedures were as described previously [30]. Briefly, flies of each genotype were sampled within a 3 hour window at age day 1 (under LD 12:12) and every alternate day starting from day 6 to day 34 (under DD). Around 8– 10 males were dissected in ice-cold PBS, treated with collagenase, fixed with 4% paraformaldehyde at room temperature, rinsed and washed with PBS containing 1% Triton-X-100. Next, blocking solution of 10% horse serum in 1% PBS-Triton-X-100 was applied for 30 min at room temperature. The following primary antibodies were applied and incubated overnight at 4uC –anti- PDF (1:20,000, rabbit, gift from Michael Nitabach, Yale University); anti-Htt (MAb2166, Chemicon,1:500); anti-elav (1:100, rat, Developmental Studies Hybridoma Bank). 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Holmes TC, Sheeba V, Mizrak D, Rubovszky B, Dahdal D, eds (2007) Circuit- breaking and behavioral analysis by molecular genetic manipulation of neural activity in Drosophila. Cold Spring Harbor, Cold Spring Harbor Laboratory Press. pp 19–52. PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11628