Preparative techniques for freezing and freeze-sectioning macrophages for energy dispersive x-ray microanalysis.

In order to study the subcellular distribution of normal intracellular electrolytes and of metal pollutants, rabbit alveolar macrophages and mouse peritoneal macrophages were maintained in standard tissue culture medium with or without various concentrations of cadmium chloride or ammonium vanadate. A variety of preparative techniques were employed to study both monolayers and cell pellets by light microscopy, transmission electron microscopy, scanning electron microscopy and energy-dispersive x-ray microanalysis. Pellets of macrophages centrifuged in narrow bore centrifuge tubes were successfully snap-frozen in liquid-nitrogen-cooled liquid propane and either sectioned on a cryoultramicrotome or freeze-substituted with 1% osmium tetroxide in acetone and embedded in Epon. Spot probes of freeze-dried, frozen thin sections for normal intracellular electrolytes such as potassium, phosphorus and sulfur showed good localization to the cells and differences between organelles. Monolayers were freeze-dried and directly embedded in Epon. When Epon thin sections of these cells and of the freeze-substituted, Epon embedded pellets were obtained with a dry knife, intracellular electrolytes such as potassium, phosphorus and cadmium could still be detected by energy-dispersive x-ray microanalysis. It is concluded that in studies using snap-freezing for element localization, maximum information is obtained with the simultaneous application of a combination of preparatory techniques.