M205 fa fluorescence microscope

Zebrafish experiments were performed at the CIRSAL (Interdepartmental Centre of Experimental Research Service) of the University of Verona, Italy. The animal protocol was approved by the Italian Ministry of Health Directorate-General for animal health and veterinary medicinal products (authorization n. 662/2019-PR of 16/09/2019). The embryos were obtained from natural spawning of nacre adults (ZFIN database ID: ZDB-ALT-990423-22), according to standard protocols [27 (link)], and staged according to Kimmel [28 (link)]. Zebrafish embryos were grown at 33°C in water containing 20-mM MSM from 2 days post fertilization (dpf). Zebrafish embryos were treated with MSM up to 1 week (experimental endpoint, 9 dpf) or 2 weeks (experimental endpoint, 16 dpf). At the end of the treatment, zebrafish embryos were euthanized and collected for molecular analyses as described below. Calcein staining was performed as previously reported [29 (link)]. Imaging was performed using Leica M205FA fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Stained areas were quantified by using the ImageJ software, as previously reported [29 (link)].
Adult zebrafish (15–20 months) were grown in water containing 20 mM of MSM for 14 weeks. At the experimental endpoint, zebrafish were euthanized and collected for staining and molecular procedures, as reported below.

Zebrafish experiments were performed at the CIRSAL (Interdepartmental Centre of Experimental Research Service) of the University of Verona, Italy, under ethical authorization n. 662/2019-PR of 16 September 2019. The embryos were obtained from natural spawning of nacre adults (ZFIN database ID: ZDB-ALT-990423-22), according to standard protocols (27). Zebrafish embryos were grown at 33 °C in water from two days post fecundation (dpf). At the experimental endpoint, zebrafish embryos or adults were euthanized and collected for molecular analyses, as described below. Imaging was performed using a Leica M205FA fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Wild type (WIK) fish lines were maintained according to established protocols (Westerfield, 2000 ). The University of Otago Animal Ethics Committee approved all zebrafish research.
An enhancer test vector ZED (Bessa et al., 2009 (link)) was used to investigate the enhancer capacity of the genomic region (Chr16: 79931421–79931980) marked by rs4077450 and rs4077451. Single-cell WIK embryos were injected with 1 nl of 30 ng/μl ZED (Bessa et al., 2009 (link)) DNA construct containing either of the minor and major fragments for the rs4077450_rs4077451 SNP region and 90 ng/μl Tol2 transposase mRNA (Kawakami, 2005 (link)). Injected embryos were screened for Green Fluorescent Protein (GFP) expression 24–120 h post-fertilization (hpf) using a Leica M205 FA fluorescence microscope.