Mouse anti pdf c7

Immunohistochemistry was performed as described previously^{57 (link)}. The following antibodies were used: mouse anti-myc (9B11, 1:500; Cell Signalling Technology), mouse anti-pdf (C7, 1:1000; Developmental Studies Hybridoma Bank) and anti-mouse Alexa Fluor 568 (1:400; Thermo Fisher Scientific). The specimens were mounted using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories). Images were captured using an FV3000 (Olympus) confocal microscope and processed using Imaris software (Bitplane). For quantification of the localisation pattern of myc-DapmaCRYA-D and myc-Drome-CRY in cell bodies of l-LN_v or s-LN_v, one- to four-day-old adult male flies were entrained to a 12-h:12-h light-dark cycle (LD; light: 200 lux) at 25 °C for 3 d. A total of 10–13 brains were immunostained and imaged at each time point (ZT 1, 5, 9, 13, 17 and 21). The areas of nucleus and cytoplasm were selected using freehand selection in Fiji, an open-source image analysis software^{58 (link)}. Selection of the regions of interests (ROIs) was performed by experimenters who were blind with respect to the genotype. The mean fluorescence intensities of the ROIs were then calculated.

Adult flies (3–5 days old) were collected and fixed at room temperature for 1 h in 4% formaldehyde diluted in PBT (PBS with 0.1% Triton X-100). The brains were dissected in PBT at indicated time points and fixed in 4% formaldehyde in PBT for 20 min at room temperature. The brains were rinsed and washed with PBT three times (15 min each) and then were blocked in 10% normal goat serum in PBT for 2 h at room temperature. The brains were incubated with primary antibody, mouse anti-PDF C7 (Developmental Studies Hybridoma Bank), diluted 1:200 or rabbit anti-PERIOD (1:1,000) in PBT overnight at 4°C. After three 15-min washes, brains were incubated with secondary goat anti-mouse Cy5 (Jackson Immuno Research) or goat anti-rabbit Alexa Fluor488 (ThermoFisher, Waltham, MA, USA) antibody at 1:200 dilution overnight at 4°C, followed by extensive washes with PBT. Finally, the brains were mounted in the VectaMount Permanent Mounting Medium. Imaging was performed on a Leica Confocal Microscope SP8 system. Confocal images were obtained at an optical section thickness of 1–2 μm.