Adsorption Staining of Freeze-Substituted and Low Temperature Embedded Frog Skeletal Muscle with Cesium: A New Method for the Investigation of Protein-Ion Interactions

A new adsorption staining method for transmission electron microscopy is described by means of which cellular adsorption sites of alkali-metal ions can be visualized in freeze-substituted and low temperature embedded biological material. The main features of this staining method are: 1) the use of Cs+ -ions which are known to accumulate in living cells like K+ -ions and 2) the removal of the staining solution from thin sections of the embedded material by centrifugal force. It is shown that sections of freeze-substituted and Lowicryl embedded frog skeletal muscle which has not been treated with chemical fixatives can be stained with electron-dense Cs+ -ions: protein sites of preferential ion adsorption are visualized. These sites are similar to those accumulating monovalent ions in living cells as had been shown previously with frozen-hydrated preparations. An observed pH-dependency of the adsorption staining is consistent with the view that the ion adsorption sites are β- and γ-carboxyl groups of cellular proteins. The results obtained so far indicate that the new method can be used to investigate weak interactions between cellular proteins and different ions by electron microscopic methods