Localization artefacts in ultracytochemical ion precipitation reactions

Abstract

The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca2+--ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg2+--ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers). As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.

Effects of vinblastine on calcium distribution pattern and Ca2+,Mg(2+)-adenosine triphosphatase in rat incisor maturation ameloblasts.

“…Previous investigations have demonstrated calcium in association with ameloblast cell membranes and various organelles, using potassium pyroantimonate cytochemistry (Eisenmann et al, 1979;Appleton and Morris, 1979;Reith and Boyde, 1985;Kogaya and Furuhashi, 1988). This technique has recognized limitations (Von Zglinicki and Punkt, 1986) but its value has been confirmed for investigating changes in cellular calcium distribution under properly controlled conditions (Suzuki and Sugi, 1989;Wick and Hepler, 1980). Experimental agents such as cobalt and colchicine have been administered to rats to determine if any ated calcium pyroantimonate deposits were markedly reduced.…”

Ultrastructural phosphatase histochemistry of submandibular and parotid salivary glands of man

“…However, caution is needed because the sites of staining by techniques for phosphatases may not be the sites of the phosphatases generating the staining (von Zglinicki & Punkt, 1986).…”

Calcium and its Localization in Human Lens Fibres: An Electron Tomographic Study

“…and Escaig, 1988 ;VonZglinicki and Punkt, 1986 ;Suzuki and Sugu, 1989 ;VanIren et al, 1979), may be due to the exceptionally high protein concentration (30-40 %) in the lens fibres. Although such matrix conditions do not favour the onset of a precipitation reaction, they are known to inhibit diffusion of the final reaction product before its precipitation and to reduce resolubilization and redistribution of once formed precipitate (VanDuijn, 1991) and consequently, they improve the localization.…”