Detailed In Situ Hybridization Protocol for Xenopus Brain

The cDNA of Xenopus Vimentin and Neuroβtubulin have been used previously 33 ,34 (link). For in situ cyp19a1, an IMAGE cDNA clone (ThermoScientific, Wilmington, DE, USA; clone ID, 6944814) was used 35 (link). Sense and antisense digoxigenin-labelled riboprobes were transcribed using the Digoxigenin RNA labelling kit in accordance with the manufacturer's instructions (Roche, Mannheim, Germany). The brain sections were processed for in situ hybridisation as described previously 36 (link). After revelation using NBT/BCIP substrate, sections were either subjected to immunostaining (see below) or directly counterstained with 4’,6-diamidino-2-phenylindole (DAPI), and mounted in Vectashield medium (Vector Laboratories, Inc., Burlingame, CA, USA). For fluorescent in situ hybridisation detection, sections were incubated in HNPP (2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate)/FastRED solution (Roche) for 2–4 h as described previously 37 (link).