Flies for brain dissection were collected at Zeitgeber time (ZT) 22 and ZT10 which correspond to high and low levels of PER protein in wild-type flies, respectively (Long et al., 2014). Whole brain mounts were made using established protocol (Long et al., 2014). Brains were incubated for 48 h in primary antibodies 1:500 chicken monoclonal anti-GFP (Aves Laboratories) and 1:10,000 pre-absorbed rabbit anti-PER, rinsed 6 times in phosphate buffered saline with 0.5% Triton-X (Fisher Scientific, Pittsburgh, PA) and incubated overnight in secondary antibodies Alexa Fluor 488 anti-chicken (1:500) and Alexa Fluor 555 anti-rabbit (1:1,000) (Life Technologies). After the final rinse, brains were mounted on microscope slides in Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, California). Images were taken with a Zeiss LSM 780 NLO scanning confocal microscope (Zeiss) with all laser parameters set for optimal signal in young fly brains at ZT22 for each genotype and then held constant while imaging young ZT10 as well as old ZT22 and ZT10 flies of the same genotype. Young and old per01 mutant flies were dissected and stained along with each genotype using the same protocol. Lack of PER staining signal in per01 mutants was used as a negative control.