Quantification of BG-mediated staining intensities. Sections from per⁺ orper⁰¹ BG/TM2males were stained with anti-β-GAL-antibody. Flies were entrained and collected under 12:12 hr LD conditions. Five (per⁺) or six (per⁰¹) flies were sectioned and stained at each time point. The staining intensities in the LNs, glia, and PRs of each fly were subjectively scored by three observers (who were unaware of either genotype or ZT of the preparations), using an intensity scale from 1 to 4. The means for each genotype are plotted as a function of ZT (see Materials and Methods, including the procedure of SEM calculation). A, Staining intensities in LNs in aper⁺ background. B,per⁰¹ background; in both genetic backgrounds, the staining intensities show circadian fluctuations, although cycling is less robust and with lower amplitude in the mutant background. C, Staining of glia in aper⁺ background. D,per⁰¹ background. Only in theper⁺ background were strong circadian fluctuations of staining intensities detectable. E, PR staining in a per⁺ background.F, per⁰¹background. Again, circadian changes in staining intensities were detectable only when per⁺ was expressed.