Mvx10 microscope

For macrophage depletion, we used Tg(mpeg1:Gal4/UAS:nfsB-mCherry) embryos expressing nfsB-mCherry under the indirect control of mpeg1 promoter. nfsB-mCherry encodes an Escherichia coli nitroreductase (NTR) fusionned to mCherry protein that converts Metronidazole (MTZ) into a toxic agent that kills the cells. Tg(mpeg1:Gal4/UAS:nfsB-mCherry) embryos were incubated in zebrafish water containing 10 mM MTZ (Sigma-Aldrich) and 0.1% DMSO at 48 hpf and 24 hr before injection with Salmonella or PBS. Treatment with 0.1% DMSO was used as a control. Depletion efficiently was assessed by imaging using the MVX10 Olympus microscope just before HBV injection. Effects of macrophage depletion on embryo survival and bacterial load during infection were analyzed at 1, 2, 3, and 4 dpi.

Adult zebrafish were maintained at 26.5 °C on a 14 h light/10 h dark cycle. Microinjection was performed on randomly separated sibling embryos at one-cell stage and embryos and larvae were staged according to development at 28 °C as described elsewhere^{66 (link)}. ASO assays were performed essentially as described in David et al.^{20 (link)}. Briefly, 5 nl of 5 ng/µl of each oligonucleotide (Supplementary Table S1) were injected, and embryos were raised up to the corresponding stage to perform RT-qPCR. For CNBP knock-down^{6 (link),7 (link)}, 5 nl containing 4 ng of CNBP translation blocking Morpholino or control Morpholino (see Supp. Table S1) were injected into the yolk.
For cnbp overexpression analysis, heterozygous Tg (XIa.Eefiai:cnbpa-EGFP) fish (http://www.zfin.org) overexpressing cnbp fused to eGFP were crossed and embryos were selected according their fluorescence under stereoscopic MVX10 Olympus Microscope equipped with a MVXTV1XC Olympus digital camera. Green fluorescent embryos were grouped and marked as cnbp OE (cnbp overexpressing embryos) while non-fluorescent embryos were collected as control ones^{6 (link)}.

Four-day post-fertilization (dpf) larvae were fixed overnight in 4% (w⁄v) paraformaldehyde (PFA) in phosphate-buffered saline 1× (PBS) at 4 °C, washed and stained as described elsewhere^{6 (link)}. Specific and control ASO microinjected embryos were observed with a MVX10 Olympus Microscope and recorded with a MVXTV1XC Olympus digital camera. Quantitative parameters were determined using the ImageJ software with a custom-made plug-in as described in Gil Rosas et al.^{7 (link)} (in detail 8 craniofacial measurements were scored: 1-Transversal Meckel length; 2-area of the inner triangle defined by the Meckel cartilage (Meckel area), 3-internal angle defined by the most anterior Meckel cartilage (Meckel angle); 4-length of ceratohyal cartilages, 5-internal angle defined by ceratohyal cartilages (ceratohyal angle), 6-length of palatoquadrate + hyosymplectic cartilages, 7-distance between the most anterior Meckel and lateral fins (cranial distance) and 8-distance between ceratohyal cartilages joint and lateral fins).

A tnfa probe was amplified from total cDNA by PCR using tnfa.55 and tnfa.58 primers (Supplementary file 1) and cloned in plasmid pCRII-TOPO. Digoxigenin (DIG)-labelled (Roche, France) sense and anti-sense RNA probes were in vitro transcribed (Biolabs, France). In situ hybridizations on whole-mount embryos were as previously described (Nguyen-Chi et al., 2012 (link)). For simultaneous detection of eGFP-F proteins and tnfa mRNA by immuno-detection and in situ hybridization, fixed and rehydrated Tg(tnfa:eGFP-F) larvae were permeabilised in ice in 100% ethanol for 5 min, then in a mixture of 50% Xylene-50% ethanol for 1 hr and in 80% acetone for 10 min at −20°C as described in Nagaso et al. (2001) (link). After washes in PBS-0.1% Tween, larvae were post-fixed in 4% paraformaldehyde (PFA) for 20 min. Subsequent steps of hybridization, washes, and staining with NBT-BCIP (Roche, France) were as previously described in Nguyen-Chi et al. (2012) (link). Next, unspecific-binding sites were saturated in PBS-1% bovin serum albumin (BSA)-1% lamb serum-10% Goat serum and larvae incubated 3 days with an anti-GFP antibody (MBL, 1/500). After extensive washes, larvae were incubated with a goat anti-rabbit antibody. Stained embryos were imaged using a MVX10 Olympus microscope with MVPLAPO 1× objective and XC50 camera and using a Zeiss Axioimager with a Zeiss 40× Plan-Apo 1.3 oil objective.

Caudal fin amputation was performed on 3 dpf larvae as described in ref. 4 (link)4 (link). The caudal fin was amputated with a sterile scalpel, posterior to muscle and notochord under anesthesia with 0.016% Tricaine (ethyl 3-aminobenzoate, Sigma-Aldrich, France) in zebrafish water. For imaging, larvae were anesthetized in 0.016% Tricaine, positioned in 35 mm glass-bottom dishes (FluroDish, World Precision Instruments, UK), immobilized in 0.8% low melting point agarose (Sigma) and covered with 2 ml of embryo water containing Tricaine. Epi-fluorescence microscopy was performed using a MVX10 Olympus microscope equipped with MVPLAPO × 1 objective and XC50 camera. Confocal microscopy was performed using an inverted confocal microscope TCSSP5 SP5 with a HCXPL APO × 40/1.25–0.75 oil and a HC PL APO 0.70 ∞(infinity) × 20 objective (Leica Microsystems, France). The 3D files generated by multi-scan acquisitions were processed using Image J (NIH).

Larvae were anesthetized and mounted as previously described [21 (link)]. Epi-fluorescence microscopy was performed using a MVX10 Olympus microscope (MVPLAPO 1X objective; XC50 camera). Confocal microscopy was performed using a confocal Leica SPE upright microscope (40x HCX APO L 0.80 W and 20x CHX APO L 0.5 W objectives) and an ANDOR CSU-W1 confocal spinning disk on an inverted NIKON microscope (Ti Eclipse) with ANDOR Neo sCMOS camera (20x air/NA 0.75 objective). Image stacks for time-lapse movies were acquired at 23–26°C every 4 min, typically spanning 50 μm at 2 μm intervals, at 1024x512 or 512x512 pixel resolution. The 4D files generated from time-lapse acquisitions were processed using Image J, compressed into maximum intensity projections and cropped. Brightness, contrast, and colour levels were adjusted for maximal visibility.