M205 fca microscope

For cilia analyses z-stacks were acquired using a Leica TCS SP5II confocal microscope with a section distance of 0.3 µm. The stacks were merged using the 3D projection tool in the Leica acquisition software of the confocal and imported into ImageJ^{70 (link)}. With the help of a Wacom tablet individual cilia were traced and the length of the trace was measured in ImageJ. In Tetrahymena, only cilia in the central portion of the cell were measured. A Zeiss Axiophot epifluorescence microscope was used to verify and analyse staining efficiency and to image ALI cultures. All zebrafish live or whole mount in situ hybridization images were acquired with a Leica M125 upright microscope and a Leica IC80 HD camera for brightfield images and a Leica M205 FCA microscope equipped with a Leica DFC9000GT sCMOS camera for fluorescence images.

For wild-type and transgenic plants, the first true leaf from 2-week-old plants and the stem from 4-week-old plants were excised and live images were taken with a fluorescence stereo Leica M205 FCA microscope. High magnification images of the trichomes on the leaves and stems were taken and the morphology of trichomes was recorded.
The fluorescence of GFP-PEN1 was detected with the excitation/emission wavelength setting: 488/505–530 nm under confocal laser scanning microscopy (ZEISS LSM 880) using inoculated and non-inoculated leaves, respectively. Images were processed and analyzed with ZEN 3.0 (black version) and Fiji ImageJ software. Quantification of the relative fluorescence intensity of the GFP-PEN1 signals was performed according to the method described previously (Qin et al., 2021 (link)). Briefly, the original images were collected with the same acquisition parameters, including laser power, pinhole, detector gains, speed, zoom factor, and resolution. Fluorescence intensity was measured with background subtracted in Fiji ImageJ software. The quantification was conducted for 30 cells per genotype. Statistics analysis was performed using one-way ANOVA with Tukey’s HSD.

Male Avil-Cre mice were mated with female RNZ mice to obtain Avil-Cre:RNZ mice. Avil-Cre:RNZ pups were sampled at P4–P6, and the TG was isolated while still attached to the cranial base by specifically transecting its peripheral and central nerve terminals. Post-dissection, the whole TG was fixed in 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) for 2 h at room temperature, and rinsed thrice with 0.05 M PB. The TG was stained overnight at 37 °C with X-Gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM magnesium chloride, 1 mg/ml X-Gal in 0.05 M PB). The following day, the TG was rinsed thrice with 0.05 M PB and post-fixed in 4%PFA in 0.1 M PB overnight. Images were acquired using the M205 FCA microscope (Leica) and DFC7000T camera (Leica).

A Discovery Hybrid Rheometer HR 20-TA instrument (USA) was used to perform tensile testing. Rheological measurements were conducted by using Anton-Paar’s Modular Compact Rheometer 302 (Austria). For the physical crosslinking measurement, a 25 mm parallel plate was used and the measurements were taken at 25 °C at 1 Hz and a strain rate of 0.1%. SEM images of the hydrogels were obtained by lyophilizing the hydrogels, and images were obtained using an Apreo 2 SEM (Thermo Fisher Scientific, USA). Conductivity was calculated by measuring resistance using a Keithley 2400 two-point probe head. Reported values reflect an average over a minimum of three to five measurements obtained for each condition. The brightfield images were taken by using a Leica M205 FCA microscope (Germany) and Leica DMi 8 microscope (Germany), and the data were analyzed with FIJI software. GIWAXS data were obtained at 1W1A Diffuse X-ray Scattering Station, Beijing Synchrotron Radiation Facility (BSRF-1W1A). The monochromatic of the light source was 1.54 Å. The data were recorded by using the two-dimensional image plate detector of Eiger 2 M from Dectris, Switzerland.

Staining of cartilage and bone was done based on the previously published protocol by Walker and Kimmel [34 (link)]. Zebrafish wild-type and mutant fish at 5, 9, 14 and 30 dpf, were euthanised with an overdose of MS-222 (300mg/L), fixed in 4% PFA and transferred to 50% ethanol. For cartilage staining, specimens were immersed in alcian blue solution (0.02% Alcian Blue 8 GX, 50mM MgCL2, 70% ethanol), and for bone staining, specimens were immersed in alizarin red solution (0.5% Alizarin Red S). For double staining of cartilage and bone, specimens were immersed in double staining solution (99% alcian blue solution, 1% alizarin red solution). After staining overnight, specimens were washed twice with 50% ethanol and then immersed in water for 2 hours before being bleached in a solution of 1.5% H202 and 1% KOH until pigmentation was removed. 30 dpf specimens were then immersed in trypsin solution (1% trypsin, 35% sodium tetraborate) for 30 minutes followed by incubation in a solution of 10% glycerol and 0.5% KOH for 1 hour. All specimens were imaged with a Leica M205FCA microscope in a solution of 50% glycerol and 0.25% KOH, followed by storage in 50% glycerol and 0.1% KOH.

Zebrafish larvae around 6 days postfertilization (dpf) were incubated in 0.3× Danieau solution containing 600 μM 2-NBDG (B6035, Apexbio, Houston, Texas, USA) for 3 h and then anesthetized and immediately imaged under M205FCA microscope (Leica, Wetzlar, Germany). Finally, the fluorescence intensity of zebrafish eye lens was used to indicate the glucose uptake according to Lee et al. (43 (link)).