Smz25 stereomicroscope

An LSM 700 confocal laser scanning microscope (Zeiss) was used for live and immunofluorescence imaging. Fish embryos and larvae were anaesthetized with a low dose of tricaine, placed in a glass-bottom Petri dish (MatTek) with a layer of 1.2% low melt agarose, and imaged using W N-ACP 20X/0.5 and W Plan-Apochromat 40×/1.0 objective lenses. Immunostained fish sections were imaged using a C Apo 40X/1.1 objective lens. For FITC-Dextran microangiography, fluorescein isothiocyanate (FITC)-Dextran, 2000 kDa (Sigma) was injected into the common cardinal vein and imaged after 10 min. An SMZ 25 stereomicroscope (Nikon) was used for brightfield images of anaesthetized fish.

The AB strain zebrafishes were obtained from China Zebrafish Resource Center. The zebrafish breast cancer xenotransplantation model was established as we previously described (15 (link), 22 (link)). Briefly, MDA-MB-231 cells were collected in free culture medium and labeled red fluorescence by 5 μM 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, Sigma-Aldrich). At 48 h post-fertilization, 200 Dil-stained MDA-MB-231 cells were suspended in 20 nl medium and injected into the perivitelline space of each embryo using a microinjector to establish the zebrafish breast cancer xenotransplantation model. The juvenile zebrafish bearing the Dil-stained breast cancer MDA-MB-231 cells were incubated in aquaculture water in 6-well plates and treated with 10 μM or 20 μM ursolic acid for 48 h. The effect of ursolic acid on breast cancer cell proliferation and metastasis in zebrafish was observed under the Nikon SMZ25 stereomicroscope.

Standard WISH procedure was performed as described previously^{78 (link)}. yap1^−/−;taz^+/− embryos from a cross between yap1^+/−;taz^+/− and yap1^+/− fish were identified by their notochord or tail phenotype (see above) and fixed with 4% PFA at 24 and 32 hpf. In parallel, WT embryos from WT fish crosses were fixed with 4% PFA at 24 and 32 hpf. Both yap1^−/−;taz^+/− and WT embryos at each developmental stage were mixed into the same reaction tube after Proteinase K permeabilization. Images were acquired with Nikon SMZ25 stereomicroscope followed by genotyping. The primers used to synthesize probes for efnb2a and mrc1a are found in Table S1.

Stage 12.5 embryos were incubated with CBX (Tocris, Bristol, UK) at 3, 5, 10, 20, 30, 50, 100, 200, and 300 μM and ENX at 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 200, and 300 μM and with MMR saline as a control. After 7 h of incubation, the embryos (stages 19–20) were washed three times with 10% MMR and kept in this solution until the tadpole stage (stage 40–45) [22 (link)]. The analysis and taking of images were conducted in a NIKON model SMZ25 stereo microscope, coupled to a brightfield Amscope camera, 1× objective (embryos stage 20) and 0.63× objective (tadpole embryos) in which the morphological effects on the neurulation process. The quantification of IC₅₀ for CBX and ENX was conducted in the ImageJ software, in which the percentage of altered phenotype (incorrect closure of the neural tube, stage 19–20) and alterations in the anteroposterior zone (stage 40–45) were analyzed. The IC₅₀ was used in the dye uptake assay (LY) and ATP quantification.

To assess neuromast quantities, larvae aged 6 dpf were submerged in 5 µg/ml DASPEI dissolved in embryo medium for 15 min as previously described (Van Trump et al., 2010 (link)). Larvae were then transferred to ice-cold water for 30–45 s then immersed in 8% methylcellulose for imaging. Images were taken using a Nikon DS-Qi2 monochrome microscope camera mounted on a Nikon SMZ25 Stereo microscope (Nikon; Tokyo, Japan). Neuromasts innervated by posterior lateral line afferents and ALL afferents were tabulated separately. To image hair cells that comprise each neuromast, larvae (5–7 dpf) were immersed in 2 µM YO-PRO1 (Invitrogen; Y3603) in embryo media for 30 min, rinsed three times in embryo media (Santos et al., 2006 (link)). Larvae were then embedded on their sides in low-melting point-1.6% agarose and imaged on a confocal microscope (Leica TCS SP5, ×63/1.2 water immersion, 200 Hz, emission: 644–698 nm). Images were processed on ImageJ (v1.48; U. S. National Institutes of Health, Bethesda, MD).

Confocal images were generated using a Leica TCS SP5 confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) with ×10 numerical aperture (NA) = 0.3 HC PL Fluotar, ×20 NA = 0.7 HC PL APO CS Imm/Corr oil, and ×40 NA = 1.3 HCX PL APO CS objectives. Three-dimensional images were collected over the full range of the signal, and a maximum projection image was created using the Leica LAS AF software. A Keyence digital microscope was used to image chromogenic sections (plan apo ×10 NA = 0.45 and plan apo ×60 NA = 1.40 oil objectives). The atrial whole mount was imaged using an SMZ25 stereomicroscope (Nikon, Tokyo, Japan).

Stereoscopic images were obtained with an M205A stereo microscope (Leica, Solms, Germany) or an SMZ25 stereo microscope (Nikon, Tokyo, Japan). DIC and fluorescent images were obtained using an Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany).