V 8 stereomicroscope

Nine wild type zebrafish (21 dpf) with SL between 4.5 mm and 9 mm were used for live staining as described by [31 (link)]. 0.003% Alizarin red and 0.0025% Calcein green stains were applied sequentially for overnight incubations using water from the fish facility. Three different conditions were tested. In the first, Alizarin red stain was applied at 21 dpf (7.7 mm SL), followed by Calcein green 5 days later (8.1 mm SL), and Alizarin red was again added after 2 days (8.6 mm). In the second group, Alizarin red was applied at 21 dpf (8.0 mm SL) followed by Calcein green two weeks later (15.0 mm SL). In the third group, Alizarin red was applied at 42 dpf (16 mm SL) followed by Calcein 3 weeks later (26.0 mm SL). Finally, fish were rinsed from the staining solution and euthanized by overdose of Tricaine. Calvaria were dissected and mounted on a glass slide for imaging using the Zeiss V-8 Stereomicroscope, a Texas Red filter for detection of Alizarin red, and a GFP filter for Calcein green.

Zebrafish embryos were obtained from mating adults, maintained and raised as described previously [15 (link)]. Zygote period cleaving eggs were transferred to six-well plastic cell culture plates filled with embryo medium E3. The eggs (20 per well) were exposed to MM-129 (10 µM), IND (200 µM), or compound combination (MM-129 + IND) for 3 h. The final volume of the medium in each well was 2 mL. DMSO was used as a MM-129 solvent, and water was used as an IND solvent. The final concentration of DMSO in the wells did not exceed the concentration above 0.1%, which means that it was within the range ensuring no harmful effects of the solvent. Embryos from the control group were incubated in an E3 medium in the presence of 0.1% DMSO. During the experiment, all embryos were observed and the moment when characteristic changes occurred in all embryos of a given group was selected as the point of triggering the effect of the test compound. Each test was independently repeated three times. Cell division and zebrafish eggs development were conducted with the use of SteREO Discovery. A V8 stereo microscope (Zeiss, Jena, Germany) was used. Photos of ongoing processes were taken once every 15 min within the first three hours of incubation.

Light microscope pictures were obtained on a Zeiss V8 stereomicroscope equipped with an IcC1 camera. Confocal images of immunostained embryos were obtained on an inverted Zeiss Spinning Disk Laser Confocal Observer Z1 using a Zeiss Plan-Apochromat 63X/1.2 W objective, or a Nikon C2 point scanning confocal microscope 40X/1.2 W objective for 48 hpf embryos. For analysis of FBMN motility, embryos were manually dechorionated and mounted in 1.2% low-melting point agarose on the coverslip of a glass bottom dish (Fluorodish; World Precision Instruments). Timelapse imaging was performed at 28.5°C using a heated stage insert. Time-lapse multiple focal plane images were obtained, with each z-stack collected every five minutes for a minimum of one hour. The acquired z-stacks were exported and analyzed using AR-Elements (Nikon) software. Embryo drift was corrected using ND alignment function. For movement analysis of FBMNs, each individual FBMN was manually traced, and the distance between centroids was tracked over multiple frames using AR-Elements software. Cell migration trajectories and instantaneous speed were measured and graphed using Prism Graphpad software.