Meta 510 laser scanning microscope

Male flies 7 days old were decapitated at Zeitgeber Time (ZT, with ZT0 = lights ON, and ZT12 = lights OFF) 1, 4, 13 and 16 under LD 12:12 conditions. Heads were fixed in 4% paraformaldehyde for 4 h, washed twice in PBS, cryoprotected in 12.5% and 25% sucrose, frozen in liquid nitrogen, and then sectioned (20 μm thickness) on a cryostat. The sections were washed in PBS for 30 min and then five times in phosphate buffer with added 0.2% Triton X 100 (PBT). Afterwards, they were incubated in a mix of 5% Normal Goat Serum (NGS) and 0.5% Bovine Serum Albumin (BSA) for 30 min. Mouse nc82 primary antibodies were added to the mix (1:25) and incubated for 48 h at 4°C. The sections were then washed six times in PBT/BSA, blocked in 5% NGS for 45 min and incubated with Cy3 conjugated goat anti-mouse secondary antibodies (Jackson Immuno Research, 1:500), overnight at 4°C. After a series of washes the sections were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta510 Laser Scanning Microscope. Confocal images of the distal lamina were analyzed using ImageJ. The fluorescence intensities of single cartridges were measured as mean gray values. GraphPad Prism software was used for statistics and making graphs. Data were analyzed using one way ANOVA Tukey’s multiple comparisons test.

To measure the L2 dendritic tree perimeter, we used 21D > mCD8::GFP (as control) and MARCM flies. Sections of the distal lamina were examined using a Zeiss Meta 510 Laser Scanning Microscope. Images were deconvolved using Huygens Professional software (Scientific Volume Imaging). Changes in the perimeter of L2 dendritic trees were examined by tracing the outline of dendrites and axons of L2 cell cross-sections. Measurements were performed using ImageJ software.

Flies were collected at ZT2 (two hours after lights-on in LD 12:12) and ZT14 (two hours after lights-off in LD12:12), their heads were fixed in 4% paraformaldehyde and brains were isolated. After washing in 0.2% phosphate buffer saline with Triton X-100 (PBST) and 30 min of blocking in Normal Goat Serum (NGS) they were incubated overnight with anti-PDF primary antibody (PDF C7 1:500, Developmental Studies Hybridoma Bank). Next, samples were washed in PBST and incubated with secondary antibodies (1:500 anti-mouse Cy3, Abcam). Whole brains were mounted in Vectashield medium (Vector) and examined with a Zeiss Meta 510 Laser Scanning Microscope.

To measure whether ROS levels were increased in mul1^A6 and park¹ mutants, 7-day-old males (N = 10) of mutants and controls were decapitated at ZT0. Brains were isolated and washed twice in PBS for 10 min. Next, the tissue was incubated with MitoSOX (ThermoFisher) for 10 min and mounted in Vectashield medium. Images were collected with a Zeiss Meta 510 Laser Scanning Microscope. The method has been described by Scialò et al. [19 (link)].

HeLa cells were seeded on glass coverslips in a 24-well plate at a density of 2 × 10⁴ cells/well. After 24 h of treatment with Dicentrine (0, 10, 25, 50 µM), cells were fixed in 4% formaldehyde in PBS (10 min at RT) and permeabilized with 0.25% Triton X-100 (5 min at RT). For immuno-labeling, cells were incubated with a mouse mAb that specifically recognizes DNA/RNA G-quadruplex structures (BG4 antibody; Absolute Antibody, Oxford, UK, #Ab00174-1.1). After 2 h of incubation at RT, cells were washed three times with PBS 1X and then incubated for an additional 1 h with an antibody anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 555 Conjugate; Cell Signaling Technology, Danvers, MA, USA, #4409S). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542), and fluorescence signals were recorded by using a Zeiss Laser Scanning Microscope 510 Meta (63× magnification) (Zeiss, Jena, Germany).
IF experiments were quantified using ImageJ (version 1.53e, National Institutes of Health, NIH, Bethesda, MD, USA). At least 25 cells were screened for each condition, and the results were expressed as the fold change of the fluorescence intensity over the negative control. Histograms show the mean ± SD of three independent experiments.