Ld lci plan apochromat 25 0.8 imm corr dic m27

For data acquisition zebrafish xenografts were imaged in a Zeiss (Oberkochen, Germany) LSM710 fluorescence confocal microscope with an objective LD LCI Plan-Apochromat 25×/0.8 Imm Corr DIC M27 (Zeiss) with 5 μm interval z-stacks. Quantification analysis was performed using Fiji/ImageJ 1.8.0 software. For tumor size, Cell Counter Plugin was used and the number of total DAPI (tumor size) = mean (3 slices: Zfirst, Zmiddle, Zlast) × total no slices)/1.5. Tumor cells were identified by both DAPI pattern and CM-DiI labelling. Mitotic figures and activated caspase 3 were quantified manually in all slices of the stack and shown in percentage in relation to tumor size (total number of tumor cells). Data normalization was completed in relation to the control of each experiment to allow direct comparison between different cell lines.

Whole-mount immunostainings of fixed embryos were performed as described previously [67 ]. The following primary antibodies were used: chicken anti-GFP (1:500; Abcam), mouse anti-Spectrin 3A9 (1:10; DSHB), guinea pig anti-Uif (1:500; [68 (link)]), mouse anti-Mega (1:20; [69 (link)]), mouse anti-Crumbs Cq4 (1:50; DSHB), rat anti-DE-cadherin DCAD2 (1:50; DSHB), sheep anti-human-matriptase (1:250; R&D Systems), rabbit anti-human-prostasin (1:250; GeneTex), rabbit anti-human-HAI-2 (1:200; ThermoFisher), and rabbit anti-mCherry (1:500; Rockland). The following secondary antibodies were used in 1:500 dilutions: goat anti-mouse IgG Alexa568, goat anti-mouse IgG Alexa488, goat anti-guinea pig IgG Alexa488, goat anti-rabbit IgG Alexa568, goat anti-rabbit IgG Alexa488 (Invitrogen), goat anti-chicken Alexa488 (Jackson Immuno Research), donkey anti-chicken DyLight549 (Jackson Immuno Research), and donkey anti-sheep Alexa568 (Invitrogen). Fluorescein-conjugated chitin-binding probe (NEB) was used in a 1:500 dilution to stain chitin. Stained embryos were mounted in ProLong Gold antifade reagent (Invitrogen). Image acquisitions were performed with a LSM780 confocal microscope (Zeiss) and a LD LCI Plan-Apochromat 25×/0.8 Imm Corr DIC M27 or a Plan-Apochromat 40×/1.4 Oil DIC M27 oil immersion or a 63×/1.3 Imm Corr DIC M27 LCI Plan-Neofluar (water) objective using standard settings.