Fv1200 laser confocal microscope

AGS cells were fixed and permeabilized as described previously12 (link). Cells were treated with primary antibodies followed by visualization with Alexa Fluor-conjugated secondary antibodies (Invitrogen). F-actin was stained with Alexa Fluor 488-conjugated phalloidin (Invitrogen) and nuclei were stained with DAPI (Wako). The focal plane of the confocal microscope was set at the cell bottom. Images were acquired using an Olympus FV1200 laser confocal microscope. Images were analysed using the Image J software (NIH)58 (link).

A fluorescence stereomicroscope (MZ16FA, Leica) equipped with a CCD camera (DFC300FX, Leica) was used to observe the whole fish (Supplementary Figs. 1d, 2). Fixed fish was examined on Olympus FV1200 laser confocal microscope with 40x silicone immersion objectives (Figs. 2f, 6c, 7c, 7f–h, Supplementary Fig. 6). All other images were acquired from live fish embedded in 0.8–1% low-melting agarose (NuSieve® GTG® Agarose, Lonza) on a Glass Base dish (IWAKI, 3010-035) with Olympus FV1200 laser confocal microscope with ×20 water immersion objective (NA1.0). For confocal imaging, fish were raised in embryonic buffer containing 0.003% (w/v) N-Phenylthiourea (SIGMA, P7629) to inhibit melanogenesis. Confocal images were acquired as serial sections along the z-axis and analyzed with Olympus Fluoview Ver2.1b Viewer and Image J, and processed for presentation with Adobe Photoshop CS6. The axon length and branching frequency were measured by Imaris Filament Tracer. Morphological analyses of CaPs were restricted to the spinal segment 14–17 before 50 hpf and to 13–17 during 56–72 hpf. A neurite with more than 5 µm of length was counted as branch.

The pCAMBIA1302-35S::GFP empty vector and pCAMBIA1302-35S::BoTCP25-GFP were transformed into Agrobacterium tumefaciens GV3101 by freeze-thaw method and transiently transfected tobacco leaves. The fluorescence signal of the BoTCP25 protein in cells was observed using an Olympus-FV1200 laser confocal microscope (Shibuya, Japan).

Approximately 30 resulting HLOs were used for visualization of bile canaliculi. The organoids were incubated in HCM containing 1 µmol/L 5-(and-6)carboxy-2`,7`-dichlorofluoroscein diacetate (CDFDA) at 37 °C for 20–30 min. Subsequently, the HLOs were washed twice with HCM without CDFDA and observed using an FV1200 laser confocal microscope (Olympus, Tokyo, Japan).

The three genes RT-PCR primers were designed using Primer Premier 5.0 software (Supplementary Table 4). The full-length CDS of PbBGLU1, PbBGLU15, and PbBGLU16 without stop codon were cloned based on genomic information and constructed into pCAMBIA1305 vectors (Clontech, Beijing, country-region China) between the XbaI and BamHI (NEB) sites which have both CaMV35S promoter and GFP gene. After electroporation of these constructions into Agrobacterium tumefaciens EHA105, using pCAMBIA1305 vector as a negative control. The infection solution was injected into the epidermis of Nicotiana tabacum leaves, after culturing in the dark for 3 days, the glass slide was made and placed under the FV1200 laser confocal microscope (Olympus Corporation, Tokyo, Japan) to observe the distribution of fusion protein.