Dn ex 8

We used image stacks of the Janelia database of the larval Drosophila CNS (wandering third instar; data by courtesy of co-author JWT). Data recording followed the protocol described in Li et al. (2014 (link)): The employed labels were “mouse antineuroglian (1:50 BP-104; Developmental Studies Hybridoma Bank), rat anti-N-cadherin (1:50 DN-Ex #8; Developmental Studies Hybridoma Bank) and rabbit anti-GFP immunoglobulin (Ig) G (1:1,000; Invitrogen A11122)”. The size of the 8 bit image stacks is about 1000 × 1400 × 100 voxels (in-plane resolution: 0.5 μm, slice thickness: 2 μm, z-step: 2 μm).

To determine the diapause rate of flies exposed to different environmental conditions as described above, the ovaries were dissected in 1× phosphate-buffered saline (PBS) under a Leica MZ8 binocular microscope. The dissected ovaries were observed under an MZ8 microscope without fixation and photographed by a Zeiss Axioplan 2 fluorescence microscope or a Leica M205FA fluorescence stereomicroscope. To observe the adult brains, the female brains at 4–7 days after eclosion were dissected in PBS and fixed in 4% paraformaldehyde for 1 hr on ice, and immunostaining was carried out using the following antibodies and dilutions: mouse anti-β-galactosidase (Z378A; Promega) at 1:1000, rabbit anti-GFP at 1:500 (598; MBL), rat monoclonal anti-DN-cadherin at 1:20 (DN-Ex#8; Developmental Studies Hybridoma Bank [DSHB], University of Iowa, Iowa City, IA), and Alexa Fluor488 anti-rabbit IgG, Alexa Fluor546 anti-mouse IgG, and Alexa Fluor647 anti-rat IgG (all at 1:200 and all from Invitrogen). Images were obtained with a Zeiss LSM 510 META confocal microscope using Zeiss LSM Image Browser software.

Brains were dissected and fixed as described by Pfeiffer et al. [73 (link)]. Brains were reacted with ChAT4B1 and DN-Ex #8 (both from Developmental Studies Hybridoma Bank) to detect ChAT and CadN), followed by incubation with Cy3 conjugated anti-mouse and Cy5 conjugated anti-rat F(ab')₂ fragments (both from Jackson ImmunoResearch). After the final wash, they were rinsed 3 × 5 min in PBS with 0.1% Triton X-100 at room temp, incubated for 2 hr with 1:500 α-Bungarotoxin, Alexa Fluor 488 conjugate (ThermoFisher Scientific) in PBS 0.1% TX-100, rinsed 3 × 5min with PBS 0.1% TX-100, mounted in SlowFade Diamond Antifade Mountant (ThermoFisher Scientific) and visualized immediately. All experiments were carried out blind.

Fly brain dissection for immunostaining and live imaging has been described (Wu and Luo, 2006 (link)). Briefly, brains were dissected in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 20 min on a nutator at room temperature. Fixed brains were washed with 0.1% Triton X-100 in PBS (PBST) for 10 min twice. After blocking with 5% normal donkey serum in PBST for 1 hr at room temperature, the brains were incubated with primary antibodies overnight at 4 °C. After PBST wash, brains were incubated with secondary antibodies (1:1000; Jackson ImmunoResearch) in dark for 2 hr at room temperature. Washed and mounted brains were imaged with confocal laser scanning microscopy (ZEISS LSM 780; LSM 900 with Airyscan 2). Images were processed with ImageJ. Neurite tracing images were generated using Simple Neurite Tracer (SNT) (Arshadi et al., 2021 (link)). Primary antibodies used included chicken anti-GFP (1:1000; Aves Lab #GFP-1020), rabbit anti-DsRed (1:500; TaKaRa #632496), rat anti-Cadherin DN (1:30; Developmental Studies Hybridoma Bank DSHB DN-Ex#8 supernatant), and mouse anti-Bruchpilot (1:30; DSHB nc82 supernatant).

After the recordings, the brains were dissected out and fixed in 4% PFA at room temperature for 30-60 mins. Quick washes with PBS were followed by incubation in PBS containing 0.2% Triton-X (PBS-T) for 1 h. Then, the brains were incubated in 5% normal goat serum at room temperature for 2–3 h or kept at 4 °C for overnight incubation. After this, the samples were incubated in 1:30 rat anti-DN-cadherin (DN-EX #8, Developmental Studies Hybridoma Bank) and 1:500 rabbit anti-GABA (A2052, Sigma-Aldrich) or 1:200 rabbit anti-Lucifer yellow (A5750, Molecular Probes) for 1–2 days at 4 °C. Brains were washed in PBS-T for several hours at room temperature and incubated with 1:500 goat anti-rat with Alexa 405 (ab175671, Abcam), 1:500 goat anti-rabbit with Alexa 633 (A21070, Molecular Probes) or goat anti-rabbit with Alexa 488 (A11008, Molecular Probes) and 1:10⁵ Streptavidin with Alexa 488 (S11223, Molecular Probes) or Streptavidin with Alexa 568 (S11226, Molecular Probes) for 1–2 days at 4 °C. Then the brains were washed multiple times with PBS and PBS-T for 3–4 h and mounted in Vectashield anti-fade mounting media (H-1000, Vector Laboratories). Morphological images were obtained using Nikon A1R MP+ microscope and NIS-C software version 5.1 (Nikon).