laevis.2. Material and Methods2.1. Animals and Tissue ProcessingAll animals were treated according to the regulations and laws of the European Union (EU Directive 2010/63/EU) and Spain (Royal Decree 53/2013) for care and handling of animals in research, after approval from the Universities of Extremadura Verdinexor (KPT-335)? and Complutense to conduct the experiments described. The number of animals used in the present study was the minimum to guarantee the correct interpretation of the results. A total of 82 Xenopus laevis embryos, larvae, and juveniles were used (Table 1). X. laevis embryos and larvae were carefully staged in accordance with Nieuwkoop and Faber [37]. Representative developmental stages are shown in Figure 1. Adult X.

laevis were purchased from commercial suppliers (XenopusOne, Dexter, MI, USA) and the different developing specimens were obtained by breeding induced by chorionic gonadotropin (Pregnyl; Organon, West Orange, NJ, USA) and kept in tap water at 20�C25��C. Young larvae were raised on Mikropan Growth Food (Sera, Heinsberg, Germany), and older larvae and juveniles were fed liver meat. At appropriate times, embryos, larvae, and juveniles were deeply anesthetized by immersion in a 0.3% solution of tricaine methanesulfonate (MS222, pH 7.4; Sigma Chemical, St. Louis, MO, USA) and used for the different sets of experiments. Specimens were fixed by immersion for 20 hours at 4��C in 4% paraformaldehyde (PFA) in PB (phosphate-buffered solution 0.1M, pH 7.4) or MEMFA (0.1M MOPS��4-morpholinopropano sulfonic acid��2mM ethylene glycol tetraacetic acid, 1mM MgSO4, 3.

7% formaldehyde). The late larvae and juveniles were perfused transcardially with 0.9% sodium chloride, followed by the same fixative solutions. The brains were dissected out and postfixed for 3 hours at 4��C.Figure 1Stereomicroscope images of selected lateral views of Xenopus laevis tailbud embryos ((a)�C(f)) and a dorsal view of a free-swimming tadpole (g) according to developmental stages (St) of Nieuwkoop and Faber [37]. Scale bars: 1mm.Table 1Number of animals investigated at different stages of development with Isl1 immunohistochemistry.Maturational aspects Batimastat in the developing X. laevis retina were examined in semithin (morphological analysis) and cryostat sections (immunohistochemical analysis). For the morphological analysis, some fixed embryos and postnatal specimens were rinsed in PB, postfixed in 2% osmium tetroxide for 2h, dehydrated in a graded series of acetone and propylene oxide, and embedded in Spurr’s resin. Serial frontal 2��m sections were cut in a Reichert Jung microtome. The sections were stained with 1% toluidine blue in 1% aqueous borax.