Alkaline phosphatase chromogen bcip nbt substrate

IHC staining was performed on sections prepared from femurs of 4-day-old mice using a ABC kit and DAB kit (Vector Laboratories, CA, USA) according to the manufacturer’s instructions, as previously described37 (link). The primary antibodies used for IHC analysis are summarized in Table S1. Methyl green was used for counterstaining. The intensity of IHC staining and the number of positively stained cells were measured and calculated using ImageJ 1.44 software following the user guide.
ISH was performed on sections prepared from paraffin-embedded samples. The RNA probes for type X collagen and indian hedgehog (Ihh) were labeled with digoxigenin (DIG) using a RNA labeling kit (Roche Life Science, IN, USA), according to the manufacturer’s instructions. The DIG-labeled RNA probes were detected by an enzyme-linked immunoassay with anti-DIG-AP antibody (Roche Life Science) and alkaline phosphatase chromogen (BCIP/NBT) substrate (Vector Laboratories) to produce a blue/purple color for positive signals.