SIMULTANEOUS LOCALIZATION OF PROTEOGLYCAN BY LIGHT AND ELECTRON-MICROSCOPY USING TOLUIDINE BLUE-O - STUDY OF EPIPHYSEAL CARTILAGE

The simultaneous localization of proteoglycan by light microscopy and EM was demonstrated by fixing rat epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy and those cut for EM required no further staining, although, in the latter case, staining with uranyl acetate and Pb improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycan is both precipitated and stained by the presence of toluidine blue O during fixation.