Localization of cations by pyroantimonate. I. Influence of fixation on distribution of calcium and sodium. An approach by analytical ion microscopy.

Abstract

A modification of the potassium pyroantimonate (PA) method for localization of calcium and sodium was tested using skeletal muscle of mouse. Massive diffusion of these cations, depending on the method of fixation, was demonstrated by analytical ion microscopy (AIM) images on the optical microscopy level. Rapid penetration of the fixative appeared to be the principal condition that reduced diffusion of Ca2+ and Na+. Paraformaldehyde (2%) appeared more efficient than glutaraldehyde (1%) for preserving metal composition. Addition of 1% phenol strikingly improved the quality of the AIM images. Supersaturated PA (4%) appeared to retain about 10 times more sodium in the tissue than insaturated PA (2%). The role of different buffers is also discussed, particularly collidine, which permitted better preservation of sodium. Fixation with this buffer should be very useful for study by AIM of large-scale distribution of sodium. These results are analyzed at the ultrastructural level in the accompanying report.

Localization of calcium changes in stimulated rat mast cells.

“…We followed a modification of the Komnick andKomnick method (1962,1963) according to Mentre and Escaig (1988). A 2 % pyroantimonate (PA) (RP, KSb(OH)6) (Merck; Darmstadt, FRG) solution was concentrated to 10% by gentle boiling.…”

“…These two complementary approaches have shown similar calcium localizations on materials differing greatly by their preparative procedure. The PA technique has been extensively used but also criticized (see Mentre and Escaig, 1988;Mentr6 and Halpern, 1988, for review). A major question that arises is whether calcium remains at its original binding site during the processing or whether it diffuses.…”

Restoration of Mature Etiolated Cucumber Hypocotyl Cell Wall Susceptibility to Expansin by Pretreatment with Fungal Pectinases and EGTA in Vitro

“…Samples were fixed according to the method from Mentré and Escaig (1988). Ultrathin sections were examined by transmission electron microscope (Hitachi 600-A-2).…”

Cytochemical Ca2+ Distribution in Activated Discoglossus pictus Eggs: A Gradient in the Predetermined Site of Fertilization

“…According to Klein et al (20) antimonate is able to precipitate Ca2+ 1 pM, Mg2+ 10 pM and Na+ 1 OmM. Standard PA fixation conditions (with Os04 as a primary fixative) are considered to selectively detect Ca2+ and to allow a considerable loss of Na+ (25,26,34). As regards the state of the Ca*+ revealed by the PA method, some authors claim it is ionic and easily exchangeable Ca2+ (20,25,26).…”

“…Standard PA fixation conditions (with Os04 as a primary fixative) are considered to selectively detect Ca2+ and to allow a considerable loss of Na+ (25,26,34). As regards the state of the Ca*+ revealed by the PA method, some authors claim it is ionic and easily exchangeable Ca2+ (20,25,26). Some authors, instead, believe that the PA method is not likely to detect either free or tightly bound Ca'+, but rather it may mainly reveal pools of easily exchangeable Ca2+ ready to be mobilized in order to act as regulator of several cell functions (reviewd in 34).…”