Circadian Clock Protein Localization

Brains from w¹¹¹⁸; + / + ; 3xHA-Nipped-A, w¹¹¹⁸, UAS-dicer; 3 × HA-Nipped-A/ + ; Nipped-A RNAi3/ + and w¹¹¹⁸, UAS-dicer; 3 × HA-Nipped-A/tim-Gal4; Nipped-A RNAi3/ + flies were collected at ZT0, dissected, fixed, immunostained and imaged by confocal microscopy as described^{62 (link)}. The primary antibodies used were anti-CLK GP50^{62 (link)} at a 1:3000 dilution, preabsorbed rabbit anti-PER antibody (a gift from Michael Rosbash) at a 1:20,000 dilution and mouse anti-HA antibody (Sigma #H9658) at a 1:1000 dilution. Secondary antibodies used were donkey anti-rabbit Alexa 488 (Jackson ImmunoResearch #711-545-152), donkey anti-mouse Alexa 488 (Jackson ImmunoResearch #715-545-150), donkey anti-Guinea pig Cy3 (Jackson ImmunoResearch #706-165-148) and donkey anti-mouse Cy3 (Jackson ImmunoResearch #715-165-150), all at a 1:200 dilution. Confocal stacks were imaged using an Olympus FV1000 confocal microscope equipped with 20 × 0.85 NA and 100 × 1.40 NA oil-immersion objectives as described^{23 (link)}.