Control of Vertebrate Skeletal Mineralization by Polyphosphates
Figure 5
Resolving polyP distribution by spectral isolation of DAPI 580 nm emission and attenuation by alkaline phosphatase (ALP) treatment.
(A) Control growth plate section; (B) a different control growth plate section (higher magnification); (C) ALP-treated (+ALP) growth plate section. First column, green: 430 nm emission (DAPI-DNA) Second column, red: 580 nm emission (DAPI-polyP). The third column is an overlay of the 430 and 580 nm images to show the spatial distribution of polyP that occurred primarily in the hypertrophic zone of control sections but was absent in ALP-treated sections. The spectral analyses in the last column show profiles for the various ROI outlined in the smaller inset image. Growth plate regions analyzed included the region above the hypertrophic matrix (red ROI), in the hypertrophic matrix (green ROI), and in the bone (blue ROI). The emission profile of each control (−ALP) hypertrophic matrix (green ROI) shows a shift to a higher wavelength emission compared to the other −ALP regions, suggesting the presence of polyP. All regions analyzed in +ALP sections showed an emission profile similar to that of DAPI-DNA, suggesting polyP was hydrolyzed from the hypertrophic matrix after exposure to active ALP. All scale bars represent 150 µm.