Bm purple substrate for alkaline phosphatase

RNA antisense probes were labeled with UTP-digoxigenin (11209256910, Roche Applied Science, Indianapolis, IN, United States) and used for whole-mount in situ hybridization (ISH) as described previously (Merino et al., 1998 (link)). Samples were treated with 60 μg/ml proteinase K for 25 min at 21°C for Bmp7, Fgfr1, Fgfr2, Fgfr3, Mkp3, Msx2, and Wif. Bambi required 70 μg/ml proteinase K for 28 min at 25°C; 60 μg/ml was used for 22 min at 21°C for Bmp4 and Dkk. Fgf8 was treated with 15 μg/ml for 20 min at 21°C. The hybridization temperature was 68°C, and post-hybridization washes were at 70°C for all genes. The signal of ISH was visualized with BM-Purple substrate for alkaline phosphatase (Roche Applied Science). Images were acquired with the Nikon Stereoscope Fluorescence Microscope SMZ1500 (Nikon Corporation, NY, United States) or in AxioZoom V.16 microscope (Carl Zeiss, Oberkochen, Germany) using Zen lite software (Carl Zeiss, Oberkochen, Germany).

RNA probes were labeled with UTP-digoxigenin (Roche Applied Science, Indianapolis, IN, United States) for their use in micromass in situ hybridization, as previously described by Chimal-Monroy et al. (2002) (link). Samples were treated with 10 μg/mL of proteinase K (PK) for 5 min at 20°C for all genes. The hybridization and post-hybridization washes were at 65°C. Signal was visualized with a BM-Purple substrate for alkaline phosphatase (Roche Applied Science). Images were acquired in AxioZoom V.16 microscope (Carl Zeiss, Oberkochen, Germany) using Zen lite software (Carl Zeiss, Oberkochen, Germany).