Fig 1

(A, B) Mouse embryos (E 10.5) were treated with Proteinase K (10 μg/ml) for 10 minutes (A) or 20 minutes (B) prior to probe hybridization with mouse antisense shh probes. (A) Shorter incubation time results in a rather superficial staining pattern in the zone of polarizing (ZPA) activity in limbs and posterior parts of the notochord. (B) Prolonged Proteinase K treatment damages the embryo integrity with loss of tissue, whereas hybridization is improved in deeper layers after in situ hybridization. (C) Safe Proteinase K treatment (10 μg/ml) in 5 dpf old zebrafish larvae without tissue disintegration indicates a staining pattern after hybridization with zebrafish specific antisense shha probe in the anterior part of the early larva (enlarged picture in C´). No signals are detected in the posterior tissues. (D) Improving tissue permeability by Trypsin digestion (10 μg/ml) prior to hybridization with antisense zebrafish shha probe is insufficient to detect shha expression in the corresponding tissues (enlargement in D´). For staining, all embryos were incubated in BM purple overnight.