Anti digoxigenin alkaline phosphatase conjugated fab fragments

The detection of human cells within the resected paraffin-embedded tumour tissues was performed as described previously [21 (link)]. In brief, deparaffinised and rehydrated sections were digested for antigen retrieval with 5 µg/mL proteinase K (Roche Diagnostics, Mannheim, Germany) for 15 min at 37 °C. Prehybridisation was completed at 42 °C for 1 h in a hybridisation buffer consisting of 4 × SSC (saline sodium citrate), 50% deionised formamide, 1 × Denhardt’s solution, 5% dextran sulfate and 100 µg/mL salmon sperm DNA. Samples were further incubated for 16 h at 42 °C with fresh hybridisation buffer supplemented with 0.2 ng/µL heat-denatured digoxigenin-labelled ALU probe, which was synthesised by PCR as described previously [19 (link)]. Detection was performed using anti-Digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche Diagnostics) and NBT/BCIP (Linaris) as substrate. Sections were counterstained with Methyl Green (Linaris), mounted with Neomount (Merck-Millipore, Burlington, MA, USA) and imaged using a Keyence BZ-X800 microscope (Keyence).

Whole-mount in situ hybridization was performed using fixed E12.0, E13.0, E14.0, E14.5 and E15.0 palates. The digoxigenin-labeled RNA probes used in this study were prepared using a DIG RNA labeling kit (Roche) according to the manufacturer’s protocol using each cDNA clone as the template. The probes were synthesized from fragments of Runx1, Shox2, Msx1, Shh, Bmp4, Tgfb3, Socs3 and Stat3 (Allen Institute for Brain Science) and were amplified with T7 and SP6 adaptor primers through PCR. After hybridization, the expression patterns for each mRNA were detected and visualized according to their immunoreactivity with anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche), as previously reported^{2 (link)}. At least three embryos of each genotype were used for each analysis.

The digoxigenin-labeled RNA probes used in this study were prepared using a DIG RNA labeling kit (Roche) according to the manufacturer’s protocol using each cDNA clone as the template.
The probes were synthesized from fragments of Runx1, Runx2, Runx3, Sox2, Lgr5, Stat3 and Amelogenin (Allen Institute for Brain Science) and were amplified with T7 and SP6 adaptor primers through a PCR. After hybridization, the expression patterns for each mRNA were detected and visualized according to their immunoreactivity with anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche), as previously reported^{9 (link)}. At least three embryos of each genotype were used for each analysis.