Ethyl 3 aminobenzoate methanesulfonate salt

Scopolamine hydrobromide 99% (Xi an Sost Biotech Co. Ltd., China), donepezil hydrochloride (Cipla EU Ltd., UK), ethanol (NedStar, Amsterdam), propylene glycol (SKC Co. Ltd., South Korea) and ethyl 3-aminobenzoate methanesulfonate salt 98% (Sigma Aldrich, USA) were purchased.

Zebrafish (Danio rerio) were maintained at the Università degli Studi di Milano according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree No 26 of the 4th of March 2014). Embryonic ages are expressed in hours post fertilization (hpf) and days post fertilization (dpf). Embryos were collected by natural spawning, staged according to Kimmel et al. [80 (link)] and raised at 28.5 °C in fish water (Instant Ocean, 0,1% Methylene Blue) in Petri dishes, according to established techniques. After 24 hpf, to prevent pigmentation, 0.003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, Saint Louis, MO) was added to the fish water. Embryos were washed, dechorionated and anaesthetized, with 0.016% tricaine (Ethyl 3-aminobenzoate methanesulfonate salt, Sigma-Aldrich) before observations and microinjection.

The number of dead zebrafish per exposure group was recorded at each measurement time point (24, 48, 72, 96, and 120 h), and the half-maximal lethal concentration (LC₅₀) was calculated. Lethality curves were established at each measurement time point. The hatching rate was also recorded. Heart rate was measured by recording heartbeat for 15 s and extrapolating to beats per minute under tricaine (ethyl 3-aminobenzoate methane sulfonate salt; Sigma-Aldrich (St.Louis, MO, USA); pH 7 adjusted by Tris to pH 9) treatment to anesthetic embryos. This measurement was repeated for three replicates per larva and the mean heart rate was used as the individual heart rate. For phenotypic or morphological analyses, zebrafish were observed and photographed at each measurement time point to check for malformations. The survival, hatching, heartbeat, and malformation rates were calculated manually.

Adult X. laevis was obtained from domestic animal venders and maintained at 20–22°C in dechlorinated water. Animals were anesthetized using 0.1% ethyl 3‐aminobenzoate methanesulfonate salt (Sigma, St. Louis, MO, MS222), pH 7.0, for surgical procedures.
Accessory limb model surgery was performed as previously described (Endo et al. 2004; Makanae and Satoh 2012). To induce ectopic blastemas, a square section of skin (1.0−1.5 × 2−3 mm) was carefully removed from the posterior side of the upper arm without damaging the underlying muscle. A ventral incision was made from the shoulder to the elbow, and the brachial nerve was dissected free and severed at the elbow level. The nerve was rerouted beneath the skin to bring the cut end to the center of the skin wound. Similarly, nerves were rerouted to the wound from the dorsal side. A square section of skin (1.0−1.5 × 1.0−1.5 mm) was removed from the anterior lower arm and grafted to the site of the skin wound.

Zebrafish (D. rerio) were maintained at the University of Milan, Via Celoria 26 – 20133 Milan, Italy (Autorizzazione Protocollo n. 295/2012-A – December 20, 2012) and Cogentech s.c.a.r.l. via Adamello 16 – 20139 Milan, Italy (Autorizzazione Protocollo n. 007894 – May 29, 2018). Zebrafish strains AB, Tg(CD41:GFP), Tg(TOPdGFP) and p53^M214K (Dorsky et al., 2002 (link); Berghmans et al., 2005 (link); Lin et al., 2005 (link)) were maintained according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree No 26 of the 4th of March 2014). Embryos were staged and used until 5 days post fertilization, a time windows in which zebrafish is not considered an animal model according to national guidelines (Italian decree No 26 of the 4th of March 2014). Embryos were staged as described in Kimmel et al. (1995) (link) and raised in fish water (Instant Ocean, 0.1% Methylene Blue) at 28°C in Petri dishes, according to established techniques. Embryonic ages are expressed in hours post fertilization (hpf) and days post fertilization (dpf). To prevent pigmentation, 0.003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, St. Louis, MI, United States) was added to the fish water. Embryos were anesthetized with 0.016% tricaine (Ethyl 3-aminobenzoate methanesulfonate salt, Sigma-Aldrich) before proceeding with experimental protocols.

Zebrafish (Danio rerio) were maintained at the University of Milan, Via Celoria 26 – 20133 Milan, Italy (Aut. Prot. n. 295/2012-A – December 20, 2012). Zebrafish strains AB, and Tg(mpeg1:mcherry) (Ellett et al., 2011 (link)), were maintained according to international (EU Directive 2010/63/EU) and national guidelines (Italian decree No. 26 of the 4th of March 2014). Embryos were collected by natural spawning, staged according to Kimmel et al. (1995) (link) and raised at 28°C in E3 fish growth medium (Instant Ocean, 0,1% Methylene Blue) in Petri dishes, according to established techniques. After 24 hours post fertilization (hpf), 0,003% 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, Saint Louis, MO, United States) was added to the fish water to prevent pigmentation. Embryos were washed, dechorionated and anaesthetized with 0.016% tricaine (Ethyl 3-aminobenzoate methanesulfonate salt; Sigma-Aldrich), before observations, microinjection and picture acquisitions.