Stereo discovery 20 microscope

Zebrafish in vivo skeletal staining was incubated with 0.05% Alizarin red (Sigma, A5533) for 1 hour and then washed with system water three times. Alizarin red and Alcian blue whole skeleton staining was performed as described 12 (link). For histological analysis, samples were fixed in 4% paraformaldehyde, decalcified in 15% EDTA and embedded in paraffin as described 32 (link). Sections (5-μm thick) were stained with Safranin O/Fast Green and hematoxylin and eosin (H & E). Picric-sirius red staining was used to detect collagen fiber density and organization as described 33 (link). Zebrafish embryos were imaged with a SteREO Discovery 20 microscope (Carl Zeiss) or LSM880NLO confocal microscope with a 20× water immersion objective (Carl Zeiss). The ceratohyal length measurements, the relative area of osterix labeled osteoblasts measurements and the mineralization intensity measurement were conducted using Image J.

Zebrafish in vivo skeletal staining was incubated with 0.2% Calcein (Sigma, C0875) solution (pH 7.0) for 10 min or 0.2% Calcein blue (Sigma, M1255) solution (pH 7.0) for 1 h or 0.05% Alizarin red (Sigma, A5533) for 1 h and then washed with system water three times. Cell internal membranes labeled with vital dye BODIPY TR Methyl Ester (C34556, Invitrogen) as described by the manufacturer, the embryos were stained with 100 μM MED for 1 h, then wash three times with egg water. BODIPY TR Ceramide (D7540, Invitrogen) prominent labeling of the Golgi apparatus with 50 μM for 2 h. LysoTracker Green DND-26 (L7526, Invitrogen) was used to in vivo label acidic notochord vacuoles after 5–6 dpf, 50 μM stained for 1–2 h depending on acidification degree of notochord vacuoles. Zebrafish embryos were anesthetized in tricaine (Sigma) and mounted in 1% LMP agarose. Calcein staining embryos were imaged with a SteREO Discovery 20 microscope (Carl Zeiss). Other live staining embryos were imaged with a LSM880NLO confocal microscope with a ×20 water immersion objective (Carl Zeiss). Images collected every 10 min for time series live cell imaging of notochord development from 20 to 30 hpf.

Sequences of respective genes that cover 0.4-1.0 kb of coding sequences were PCR-cloned. Later, PCR products were cloned into the pGEMT-easy vector (Promega). Digoxigenin-labeled probes were generated by in vitro transcription (DIG RNA Labeling Kit, Roche, America) and ISH was carried out according to the zebrafish book. Briefly, embryos were fixed overnight at 4 °C in 4% paraformaldehyde (PFA), dehydrated through a methanol series and stored overnight at − 20 °C. Subsequently, the embryos were rehydrated in PBST (PBS + 0.1% Tween 20) and pre-hybridized for 2–5 h at 68.5 °C in hybridization solution containing 50% formamide. Hybridization was performed with 0.5-1 ng/µl antisense probes diluted in hybridization solution overnight at 68.5 °C. This was followed by washing with 2× SSCT buffer and blocking in 1% blocking reagent (#11,096,176,001, Roche, America) at room temperature for 1 h. The embryos were then incubated with anti-Dig-AP antibody (dilution 1: 5000, #11,093,274,910, Roche, America) at 4 °C overnight. Embryos were rinsed extensively for 3 h, and then stained with NBT/BCIP solution (dilution 1: 50, #11,681,451,001, Roche, America). The WISH images were captured using a SteREO Discovery 20 microscope (Carl Zeiss, Germany). The primers used for ISH were designed using SnapGene and checked by Oligo 7. Relevant primer sequences are listed in the Supplementary Table 1.