Sp5 confocal microscope

Drosophila embryo collection, fixation, and antibody staining were carried out as previously described [15 ]. The following antibodies were used: FITC-conjugated goat anti-HRP (Jackson ImmunoResearch #123-095-021, 1:100), Alexa 647-conjugated goat anti-HRP (Jackson #123-605-021, 1:100), mouse anti-Fasciclin II (Developmental Studies Hybridoma Bank [DSHB] #1D4, 1:100), mouse anti-βgal (DSHB #40-1a, 1:150), mouse anti-Robo3 (DSHB #15H2, 1:100), mouse anti-HA (Covance #MMS-101P-500, 1:1000), rabbit anti-c-Myc (Sigma-Aldrich #C3956, 1:500), Cy3-conjugated goat anti-mouse (Jackson #115-165-003, 1:1000), Alexa 488-conjugated goat anti-rabbit (Jackson #111-545-003, 1:500). Embryos were genotyped using balancer chromosomes carrying lacZ markers. Ventral nerve cords from embryos of the desired genotype and developmental stage were dissected and mounted in 70% glycerol/PBS. Fluorescent confocal stacks were collected using a Leica SP5 confocal microscope and processed by Fiji/ImageJ [16 (link)] and Adobe Photoshop software.

Mouse embryos were staged by designation the noon of the day when the vaginal plug was observed as embryonic day 0.5 (E0.5). Embryos of desired age were disected, fixed in 4% paraformaldehyde (PFA) from 45 minutes up to 4 hours at 4°C, washed with PBS, cryopreserved in 30% sucrose and frozen in OCT (Sakura). The cryosections (10–12 μm) were permeabilized with PBT (PBS with 0.1% Tween), blocked with 10% BSA in PBT and incubated with primary antibody (1% BSA in PBT) overnight at 4°C. Sections were washed with PBS, incubated with fluorescent secondary antibody (Life Technologies, 1:500) for one hour at room temperature, washed with PBS, counterstained with DAPI and mounted in Mowiol. The images were taken on Leica SP5 confocal microscope and were processed (contrast and brightness) with Adobe Photoshop. For hematoxylin-eosin staining, embryos were fixed in 8% PFA overnight, processed, embedded in paraffin, sectioned (8 μm), deparaffinized and stained. For β-galactosidase staining, embryos were fixed in 2% PFA, washed with rinse buffer (0.1 M phosphate buffer pH 7.3, 2 mM MgCl₂, 20 mM Tris pH 7.3, 0.01% sodium deoxycholate, and 0.02% Nonidet P-40) and incubated in X-Gal staining solution (rinse buffer supplemented with 5 mM potasium ferricyanide, 5 mM potassium ferrocyanide, 20 mM Tris pH 7.3, and 1 mg/ml X-gal) at 37°C for 2 hours and at room temperature overnight shaking.

Images were acquired with a Leica SP5 confocal microscope, analyzed utilizing Imaris and ImageJ, and processed with Adobe Photoshop and Adobe Illustrator. 3D images of fixed samples were taken with a 40×/1.3 numerical aperture (NA) oil immersion objective. 4D in vivo images were obtained at ∼20°C. To analyze rotation, a 20×/0.7 NA oil immersion objective and Leica hybrid detectors (standard mode) were used, with time points every 2–4 min for 1–6 hr.
Transmission electron microscopy protocols and atomic force microscopy measurements are detailed in the Suppl. Information section.

Drosophila embryo collection, fixation and antibody staining were carried out as previously described [40 (link)]. The following antibodies were used: FITC-conjugated goat anti-HRP (Jackson #123-095-021, 1:100), mouse anti-Fasciclin II (DSHB #1D4, 1:100), mouse anti-βgal (DSHB #40-1a, 1:150), mouse anti-Robo1 (DSHB #13C9, 1:100), rabbit anti-GFP (Invitrogen #A11122, 1:1000), mouse anti-HA (Covance #MMS-101P-500, 1:1000), Cy3-conjugated goat anti-mouse (Jackson #115-165-003, 1:1000), Alexa 488-conjugated goat anti-rabbit (Jackson #111-545-003, 1:500). Embryos were genotyped using balancer chromosomes carrying lacZ markers, or by the presence of epitope-tagged transgenes. Ventral nerve cords from embryos of the desired genotype and developmental stage were dissected and mounted in 70 % glycerol/PBS. Fluorescent confocal stacks were collected using a Leica SP5 confocal microscope and processed by Fiji/ImageJ [39 (link)] and Adobe Photoshop software.

Primary antibodies used were: Rabbit Zeb1 Proteintech 21544-AP) 1/100 and Mouse YAP (Santa cruz sc-101199) 1/100. Secondary antibodies were from Invitrogen, and used at 1:500 for 2 hr at room temperature along with DAPI. Cell culture samples were imaged with a Leica SP5 confocal microscope using a 63x oil immersion objective and processed using Adobe Photoshop. Cells were assessed over three independent experiments counting 200–300 cells per condition.