CLK GP50 antibody does not cross-react with DAC and detects CLK IR only in oscillator cells. (A) Western blots containing 1.0 μl and 5.0 μl of in vitro transcribed and translated DAC (IVTT DAC), 0.1 μl and 1.0 μl of in vitro transcribed and translated CLK (IVTT CLK), and 100 μg of head extract from flies collected at ZT14 probed with either DAC monoclonal antibody (mAbDAC) (left) or CLK GP50 antiserum (GP50) (right). (B) Wild-type L3 larval brains were dissected and fixed at ZT23, immunostained with CLK and PER antibodies, and imaged by confocal microscopy. Images show an 18 μm Z-series projection of the CNS, where dorsal is at the top. CLK GP50 (left panel), PER (Middle panel) or merged CLK GP50 + PER (right panel) immunostaining in DN1* neurons (brackets) and DN2 or s-LNv neurons (arrowheads) in both brain hemispheres. DN1*, s-LNv, and DN2 neurons are as defined in the legend for Fig. 2. Co-localization of CLK (red) and PER (green) is shown as yellow. All images are representative of three or more independent experiments.