Glycogen, its chemistry and morphological appearance in the electron microscope.11. The complex formed in the selective contrast staining of glycogen.

Selective contrast staining of glycogen in untreated ultrathin sections of aldehyde-fixed tissues, double-fixed with 1% osmium tetroxide containing 0.05 M K3Fe(CN)6 as reported previously (De Bruijn, 1973), may also be obtained by the addition of either K4Fe(CN)6,K3Co(CN)6,K2Ru(CN)6, or K4Os(CN)6. On the other hand, addition of K3Cr(CN)6, K2Ni(CN)4, K3Mn(CN)6, K3Rh(CN)6, K2Pd(CN)4, K2Pt(CN)4, or K3Ir(CN)6 produces no effect. Hexavalent osmium oxide compounds, such as K2OsO4 and OsO3-2 pyridine, react selectively with a native (or acquired) ligand in the aldehyde-fixed glycogen, but do not render it more electron dense than its immediate surroundings. The presence of these osmium oxides is detected and they are rendered more electron dense by an accumulation reaction by the application on ultrathin sections of phosphotungstic acid (PTA) or a mixture of K2OsO4 and K4Fe(CN)6. As selective contrast staining of glycogen is also obtained by double fixation of the aldehyde-fixed tissue with 0.05 M K2OsO4 solutions containing 0.05 M K4Fe(CN)6 or 0.05 M K4Os(CN)6, it is postulated that in such tissue, both the selective reaction of K2OsO4 with the ligand in the aldehyde-fixed glycogen, and the accumulation of heavy metal at the sites occupied by the K2OsO4, occur simultaneously. A proposal for the constitution of this heavy metal osmium/cyanide complex is formulated and arguments are presented that both compounds are formed in the selective contrast stained glycogen areas of such treated tissues. The relative contribution of the components to the final contrast and its complex character is demonstrated by staining ultrathin glutaraldehyde sections intermittently with 0.05 M solutions of K2OsO4 and K4Fe(CN)6; it is shown that after at least three intermittent reactions with both K2OsO4 and K4Fe(CN)6, the glycogen areas in such sections became contrast stained.