For colorimetric in situ hybridization embryos were treated with 10μg/mL proteinase K (Invitrogen 25530015) for 30 minutes, post-fixed, pre-hybridized at 68°C for 1hour and hybridized with 500ng/mL digoxigenin-labeled riboprobes at 68°C overnight as previously described (Lewandowski et al., 2015 (link)). BM purple (Sigma-Aldrich 11442074001) staining was visualized in dissected forelimbs using a table-top Leica light microscope equipped with a DFC420C Leica camera.
Fluorescent in situ hybridization was conducted using HCR v3.0 reagents as previously described (Ramachandran et al 2022). Briefly embryos were fixed overnight at 4°C in 4% PFA/PBS and rehydrates with MeOH/PBST (0.1% Tween-20). Embryos were then treated with 10μg/mL proteinase K for 30 minutes, incubated in 4nM probe overnight at 37°C, and then in 60pmol hairpin overnight at room temperature. After hairpin incubation, the samples were washed and counterstained in DAPI (1:5000 dilution; Life Technologies D1306), embedded in low-melt agarose and cleared in CeD3++ as described (Anderson et al., 2020 (link)). Samples were imaged on a Nikon W1 spinning disk confocal.
Fluorescent in situ hybridization was conducted using HCR v3.0 reagents as previously described (Ramachandran et al 2022). Briefly embryos were fixed overnight at 4°C in 4% PFA/PBS and rehydrates with MeOH/PBST (0.1% Tween-20). Embryos were then treated with 10μg/mL proteinase K for 30 minutes, incubated in 4nM probe overnight at 37°C, and then in 60pmol hairpin overnight at room temperature. After hairpin incubation, the samples were washed and counterstained in DAPI (1:5000 dilution; Life Technologies D1306), embedded in low-melt agarose and cleared in CeD3++ as described (Anderson et al., 2020 (link)). Samples were imaged on a Nikon W1 spinning disk confocal.