Lci plan neofluar 25x 0.8 imm korr dic m27 objective

The CNS, CPS and attached pharyngeal nerves of larvae with the genotype OK371-Gal4/UAS-mCD8::mRFP;UAS-PaGFP(A206K)/+ were placed with ventral side down onto a Poly-L-lysine (Sigma Aldrich) coated cover slide and mounted upside down in Ringer solution. Images were acquired using a laser scanning microscope (Zeiss LSM 780) with a Zeiss LCI “Plan-Neofluar” 25x/0.8 Imm Korr DIC M27 objective. To photoactivate the GFP we used a Ti:Sapphire Chameleon Ultra II Laser (Coherent) tuned to 820nm. Laser power was set to 8% for activation of the GFP in nerves. Bleach period of 8s was sufficient to activate the GFP in axons of the nerves. This was repeated two times at different areas of the nerve. Following settings were used: image size at 161.31x161.31μm, resolution at 256x256, pixel dwell 2μs, speed 11 and region of interest (ROI) size at 140μm.
After successful activation of GFP in the nerve, low GFP signal in somas in the SEZ were visible. Those were subsequently activated using the same protocol, adjusting ROI sizes to somata. This procedure was done for AN, MN and PaN. Z-stacks were acquired using 488nm laser and 561 nm laser as excitatory sources. Fluorescence was collected using photomultiplier tubes after filtering using the MBS 488/561/633 emission filter.

Immunofluorescent images were acquired with an LSM700 confocal laser scanning microscope equipped with an LCI Plan-Neofluar 25x/0.8 Imm Korr DIC M27 objective (Zeiss, Göttingen, Germany). In the majority of cases, four random images were obtained at a 250× magnification, sampling a total tumor area of generally 1.0 mm², comprising vital areas of both tumor epithelium and stroma. Double or triple positivity of cells as well as the tumor epithelium and stroma area were determined using the overlay tool in the LSM Image Browser software (version 4.2.0.121, Zeiss). The numbers of single, double and triple positive cells were scored in the tumor epithelium and tumor stroma separately using the open source image processing program ImageJ version 1.47 (http://rsb.info.nih.gov/ij). Cells within blood vessels or lumina were not counted.

Larvae were dissected as described for extracellular recordings. A jelly pool was used to isolate the nerve of interest. Saline level was lowered until the fluids inside and outside the jelly pool were separated from each other. The nerve was cut within the jelly pool and saline was replaced with tetramethylrhodamine-dextran solution (Tmr-D) (Life Technologies, 3000MW anionic at 10mg/ml in distilled water). The preparations were stored at room temperature for 3 hr. After uptake of the dye, preparations were fixed in 4% paraformaldehyde (PFA) for 40 min, washed in PBS and mounted in Mowiol. The CNS was scanned the following day using a Laser scanning microscope (Zeiss LSM 780) equipped with a Zeiss LCI “Plan-Neofluar” 25x/0.8 Imm Korr DIC M27 objective. For colocalization with glutamatergic neurons we used larvae with the genotype OK371-Gal4/+;UAS-GCamP6s/+. Using GCamP6s for scanning the CNS had the advantage that the strong fluorescent signal was evenly distributed throughout the cells and their dendritic arborizations.
After verification of expression of the CDM motor neurons in the CNS of larvae (+/+;MT11-Gal4/10xUAS-mCD8::GFP), anterograde fillings of the left AN were done using the same procedure as described for retrograde dye filling.