Sylgard dishes

Protocols for cell isolation and library preparation were adapted from protocols developed for analysis of the Drosophila larval VNC [58 (link)]. 5-day old Ae. aegypti larvae were sorted under a fluorescent stereomicroscope to identify larvae carrying both brp-T2A-QF2w and QUAS-mcd8GFP and incubated on ice to immobilize larvae prior to dissection. Larvae were pinned dorsal side up on sylgard dishes (Dow corning) using minutien pins (Fine Science Tools) containing ice-cold PBS, filleted along the dorsal midline, and VNCs were isolated from surrounding tissue under a fluorescent stereomicroscope and transferred to 1.5 ml Eppendorf tubes containing dissociation buffer (450 μl PBS supplemented with 1% BSA solution (Sigma) and 1 mg/mL collagenase type I (Fisher Scientific, 17-100-017)). A total of 50 VNCs were dissociated (15–20 VNCs in each tube) at 37 °C with mechanical agitation (mixing at 1000 rpm with manual trituration every 10 min). Dissociated cell suspensions were filtered through 70 μm nylon filters to remove cell debris, stained with propidium iodide (PI) to label dead cells, and GFP positive/PI negative neurons were captured using a FACSArial II (BD Biosciences, San Jose, CA). 61,646 single cells were collected and resuspended in 50 μl PBS + 1% BSA solution after centrifuging for 10 mins at 300x g and 4 °C.

Ae. aegypti larvae were pinned dorsal side up on sylgard dishes (Dow corning) containing ice-cold PBS, filleted along the dorsal midline, and pinned open to expose the VNC. Intestines, fat bodies, and trachea were removed, samples were rinsed in PBS, and subsequently fixed for 30 min in PBS + 4% formaldehyde (PBSFA) on a rotary shaker. Following fixation, samples were washed twice with PBS for 2 min each and subsequently permeabilized PBST (PBS + 0.3% Triton X-100) for 15 min (3 washes, 5 min each). Specimens were blocked for 15 min in PBST containing 5% NGS (Jackson Immunoresearch) and incubated in primary antibody diluted in blocking buffer at 4 °C overnight. The following day, samples were washed twice with PBS for 2 min each, three times with PBST for 10 min each, and incubated with the secondary antibody and Hoechst (1:500) at room temperature for 1–2 hours in blocking buffer. Following staining, samples were washed with 800 μl PBST 3 times for 10 min each at room temperature, rinsed twice in PBS, and mounted in Fluoromount G mounting medium (Electron Microscopy Sciences). Slides were dried at room temperature for a minimum of 1 hr. prior to imaging. Details on antibody sources and dilutions are provided in Table S1.