MICRO-PIXE QUANTIFICATION OF HISTOCHEMICAL REACTIONS

The proton induced X-ray emission in combination with a proton microprobe (micro-PIXE) was used for the quantitative determination of a locally bound histochemical reaction end-product. The determination of proteoglycans (PG) content in the cartilage is described in detail. The cartilage from the femur and the skull of 17-19 days old embryos and from ribs of mature CBA strain mice was investigated. The 8 μm cryomicrotome sections, placed on a formvar backing, were incubated in colloidal iron or in alcian blue (dye molecule contains Cu) solutions. The determination of PG contents was made indirectly by assessment of the Fe or Cu concentrations. Differences in the PG contents were observed within all investigated cartilage types. The embryonal cartilage contains more PG than that obtained from mature animals. For the cartilage from the femur the highest content of PG was found in the mineralization front while fully formed bone trabeculae exhibited a PG level 10 times lower. In the case of intramembranaceous ossification the content of PG was also higher in the skull cartilage than in the loose connective tissue. On the basis of the performed studies some methodological conclusions can be drawn. For PG determination, the alcian blue method is more reliable than the colloidal iron technique. The review of the histochemical procedures which may be combined with the micro-PIXE method is also given. © 1990.