(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.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages: (3 dpf: A—C; 5 dpf: D—F; 7 dpf: G—I; 9 dpf J–L; 10 dpf: M–O) and illustrated in lateral (A, D, G, J, M), dorsal (B, E, H, K, N) and frontal views (C, F, I, L, O). For staining, all embryos and larvae were incubated in BM purple for 22 hours.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages (3 dpf: A—C; 5 dpf: D—F; 7 dpf: G—I; 9 dpf J–L; 10 dpf: M–O) and illustrated in lateral (A, D, G, J, M), dorsal (B, E, H, K, N) and frontal views (C, F, I, L, O). For staining, all embryos and larvae were incubated in BM purple for 22 hours.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages (1 dpf: A—C; 4 dpf: D—F; 10 dpf: G—I; 24 dpf: J–L, enlarged in J´- L´) and illustrated in lateral (A, D, G, J, J´), dorsal (B, E, H, K, K´) and frontal views (C, F, I, L, L´). Brass wild type line embryos (A—I) and casper line larvae (J–L, J´- L´) have been used. Embryos at 4 dpf (D—F) and 10 dpf (G—I) were treated with PTU to suppress pigmentation, embryos at 1dpf (A—C) and 24 dpf (J–L, J´- L´) were untreated. For staining, all embryos and larvae were incubated in BM purple for 22 hours.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages (1 dpf: A—C; 4 dpf: D—F; 10 dpf: G—I; 24 dpf J—L, enlarged in J´- L´) and illustrated in lateral (A, D, G, J, J´), dorsal (B, E, H, K, K´) and frontal views (C, F, I, L, L´). Brass wildtype line embryos (A—I) and casper line larvae (J–L, J´- L´) have been used. For staining, all embryos and larvae were incubated in BM purple for 22 hours.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages (1 dpf: A—C; 4 dpf: D—F; 10 dpf: G—I; 24 dpf J—L, enlarged in J´- L´) and illustrated in lateral (A, D, G, J, J´), dorsal (B, E, H, K, K´) and frontal views (C, F, I, L, L´). Brass wildtype line embryos (A—I) and casper line larvae (J—L, J´- L´) have been used. For staining, all embryos and larvae were incubated in BM purple for 22 hours.

Whole mount in situ hybridization was performed in embryos and larvae at different developmental stages: (0.8 dpf: A—C; 1 dpf: D—F; 4 dpf: G—I; 10 dpf: J—L; 24 dpf: M–O, enlarged in M´- O´) and illustrated in lateral (A, D, G, J, M, M´), dorsal (B, E, H, K, N, N´) and frontal views (C, F, I, L, O, O´). Brass wild type line embryos (A—L) and casper line larvae (M–O, M´- O´) have been used. For staining, all embryos and larvae were incubated in BM purple for 22 hours.