| Abstract: | Purified rat peritoneal mast cells contained 3.3 × 10?5 gm SO4 and 2.2 × 10?8 gm Ca/106 cells. The molar ratio of S/Ca in the whole cell was 600:1. Frozen thin sections of unfixed mast cells contained only sulfur (S) in the granules when examined by X-ray energy dispersive spectroscopy (EDS). Mast cells fixed in 3% glutaraldehyde and 1.5% formaldehyde in 75% ethanol (Et/Ald) or in mixed buffered aldehydes and embedded in Epon 812 or the low viscosity resin diepoxyoctane (DEO) contained S in all granules and Ca in some of the granules measured. Neither element was found in the nucleus, cytoplasm, or resin. Isolated, Et/Ald fixed and embedded granules also contained S. The presence of Ca in the granules was artifactual in that the Ca was absorbed from water in the trough of the diamond knife and/or from the filter paper used to blot the sections dry. This phenomenon was investigated further. Sections of Et/Ald fixed and embedded mast cells were incubated with 5 × 10?6 to 10?2 M CaCl2. Ca was detectable in 100% of the granules incubated at concentrations ≥ 10?4 M and reached a constant S/Ca ratio of 2.0 at concentrations ≥ 10?3 M. Ca was not detectable in the nucleus, cytoplasm, or resin at 10?2 M. A plot of S versus Ca counts from the granules of cells incubated with 10?2 M CaCl2 was linear with a slope of 2.0 and a correlation coefficient of 0.88. Et/Ald fixed cells incubated with distilled H2O had fewer granules containing Ca, 10%, than unincubated cells, 77%. Further, H2O removed all Ca from Et/Ald fixed cells embedded in DEO. These studies show that S, which is present as SO4 on the proteoglycan heparin, is readily detectable by X-ray EDS in fixed and embedded cells. An artifact of the technique is that weak anionic sites, which are most probably carboxyl groups on the proteoglycan, can bind the divalent cation Ca and cause spurious localization. |
