Plan apochromat 40 1.4 oil dic m27

Images were acquired with a LSM780 confocal microscope (Zeiss) using a 40×/1.4-NA oil objective (Plan Apochromat 40×/1.4 oil DIC M27) and appropriate laser lines set in single tracks and pixel resolution of at least 2048 × 2048. All lasers were used at 2% power or less. Images were acquired in 12-bit or 16-bit mode as z stacks with optical slice thickness set to optimal and the detector gain in each channel adjusted to cover the full dynamic range but to avoid saturated pixels. Maximum intensity projections were created in Zen10 software or in Fiji/ImageJ. For visualization, linear adjustments of the single channels for brightness over the whole image were performed. Conditions were kept constant within an experiment.

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.

Live young adult worms were placed on a 2% agarose pad containing 0.25 mM levamisole in NaCl to immobilise the worms. Images were acquired using a confocal laser scanning microscopy: Zeiss LSM780 and its acquisition software Zen with a Plan-Apochromat ×40/1.4 Oil DIC M27 objective with a zoom 2–4, a Plan-Apochromat ×63/1.40 Oil DIC M27 with a zoom 1. Spectral imaging combined with linear unmixing was used in most confocal images to separate the autofluorescence of the cuticle.