M205 fca stereo microscope

For all motility experiments, thrashing of animals were scored on day 1, day 5, and day 9 animals grown on either 2% or 4% RNAi plates from L1. Animals were collected using M9 and placed onto an NGM plate without bacteria and 10 s video recordings were taken using a Leica M205FCA stereomicroscope. Thrashing was manually recorded wherein a single body bend in one direction is scored as a single thrash and the total number of thrashes for a 10 s period are calculated. Thrashing assays were performed on three biological replicates and statistics were calculated using Mann-Whitney testing using Prism 7 software.

Zebrafish were handled according to the vertebrate animal handling protocol. AB (wild-type) zebrafish were used for all experiments. For morpholino experiments, 2-cell-stage zebrafish were injected with 1–2 nl 500 µM p53-morpholino (5′-GCGCCATTGCTTTGCAAGAATTG-3′, GeneTools PCO-ZebrafishP53-100). Non-injected embryos were used as control.
At 6 hpf, zebrafish were treated with TG003 or 2-deoxy-D-glucose (2DG, Sigma-Aldrich, # D8375-1G). Water quality conditions were: dissolved oxygen = 6.50±0.25 mg/L, salinity = 0.0 practical salinity unit (PSU) pH 7.2, conductivity = 520µS/cm), temperature = 28°C. Drug was washed out at 24 hpf (i.e.18 hours after treatment) and no additional drug was added. Water was changed every subsequent day, and fish were collected at 5 days post fertilization (dpf) for cartilage staining.
Fish were fixed, bleached and stained with Alcian Blue as described previously (Dingerkus and Uhler, 1977 (link); Sakata-Haga et al., 2018 (link)). Fish were imaged using a Leica M205 FCA stereo microscope, using the same settings for all samples (bright-field, exposure = 1 ms). Lengths of craniofacial structures were analyzed using Fiji, by an investigator to whom the treatment groups had been anonymized.

We used strains containing a translational GFP reporter of DAF-28 to quantify its expression in the head of the arrested L1 larvae. We took aliquots of 10 μl from suspensions of L1s arrested to the indicated densities after 1 or 5 days of arrest and we placed them on a microscope slide with 1 μl of 10 mM Levamisole to paralyze the animals. We then acquired pictures of these worms using a Leica M205 FCA stereo microscope. We analyzed those pictures with FIJI software, which allowed us to measure the signal intensity of a selected area. We analyzed more than 20 animals per condition and experiment, and we performed a minimum of three individual biological replicates per experiment. Due to discrepancies in the raw data between replicates, we normalized the signal measured for each worm to the average signal of the replicate. We used the averages of the normalized signal per condition and replicate to perform the statistics analysis.

For whole mount studies, scales were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS, pH = 7.8) at 4 °C for 24 h. Subsequently, three different kinds of staining experiments were respectively carried out on the scales of O. mykiss from four sample groups (CG, 7D, 14D and 21D). Each group was consisting of three fish in parallel, and six scales of each fish were used in parallel for dyeing. Scales were stained for activity of TRAcP and ALP, according to the instruction of Tartrate-Resistant Acid Phosphata stain Kit (D023–1) and BCIP/NBT stain Kit (I023–1) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) respectively.
In addition, samples were stained for presence of calcium phosphates using Von Kossa′s Kit (G3282) (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), following the manufacturer’s instructions. Breifly, scales were incubated with 5% silver nitrate under bright light for 1 h, subsequently rinsed with tap water for 1 min, developed for 5 min in 5% sodium thiosulfate, and finally rinsed thoroughly with tap water. All samples were photographed under a Leica M205 FCA stereo microscope (Leica microsystems, Wetzlar, Germany) and the stained area of Von Kossa′s stained scales was quantified with Photoshop 2019 (Adobe, San Jose, USA).

To visualise ciliated cells, trachea specimens derived from Foxj1-GFP mice were fixed in 4% PFA in PBS at 4°C for 4 h. Tiled images were acquired using an LSM 710 confocal microscope (Carl Zeiss, Jena, Germany).
For vascular imaging, the mice euthanised by administering CO₂ were intracardially perfused with 9.25% (w/v) sucrose followed by perfusion with the fluorescent dye DiI and fixed with 4% PFA in PBS. The images were acquired using the fluorescent microscope Zeiss Axio Zoom.V16 (Carl Zeiss, Jena, Germany).
To visualise BCs, ciliated cells, and nerve fibers, the trachea specimens from C57BL/6J mice fixed with 4% PFA in PBS at 4°C for 4 h were blocked by treating with 10% donkey serum, 3% BSA, and 0.1% Triton X-100 in PBS, and were then treated with primary antibodies (mouse acetylated tubulin antibody, 1:1000, T7451, Sigma-Aldrich, St. Louis, MO, USA; goat Krt13 antibody, 1:500, ab79279, Abcam; mouse β-III tubulin antibody, 1:1000, T8660, Sigma-Aldrich, St. Louis, MO, USA). After subsequent treatment with secondary antibodies (Alexa Fluor488, Alexa Fluor 555), images were acquired using a Leica SP8 confocal microscope and Leica M205 FCA stereomicroscope (Leica Microsystems, Wetzler, Germany).