Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography.

Abstract

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

“…The bulk of [3H]arachidonate was incorporated into phospholipids. For example, in HSDM~Ct cells after 2 h of incubation, ~94% of radioactivity taken up was incorporated into phospholipids and 4% was found in neutral lipids as determined by thin layer chromatography (13). These results for arachidonate incorporation into HSDM~Cj and EPU-1B cells are consistent with those previously reported (15,19,31).…”

“…As shown in Table I, 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. Nearly half of the radioactivity extracted during processing was neutral lipid; no single species of phospholipid was selectively extracted (13). Source-grain pair mask data from en face sections of each cell type were pooled to give greater accuracy in surface area determination.…”

“…Tissue Processing for EM: During processing, cells remain attached to the culture dishes. Cells were fixed in 2% glataraldehyde plus 1.5% tannic acid, postfixed with 2% osmium tetroxide, stained en bloc with 0.5% uranyl magnesium acetate, dehydrated in ethanol, and infiltrated with Epon resin as previously described (13). In selected experiments, the amount of radioactivity removed from cells during each processing step and retained by cells at the completion of processing was assessed with liquid scintillation spectrometry, as previously reported (13).…”

Transporter-Mediated Nuclear Entry of Jasmonoyl-Isoleucine Is Essential for Jasmonate Signaling

“…To evaluate the relative distribution of the tracer in the nucleus and cytoplasm, we performed electron microscopic autoradiography analyses as previously described (Farquhar et al, 1978;Krueger et al, 1985;Dashek, 2000). The callus cells were incubated with 0.4 mM 3 H-JA (10 mCi/nmol, American Radiolabeled Chemicals) in 100 mM potassium phosphate buffer (pH 5.8) for 2 h and fixed in 4% glutaraldehyde in 100 mM potassium phosphate buffer for 3 h. The samples were then postfixed in 1% osmium tetroxide for 2 h. After three rinses in potassium phosphate buffer, the cells were rapidly dehydrated in single changes of 30%, 50%, 70%, 80%, 90%, and 100% acetone.…”

Freeze Drying Method with Gaseous Nitrogen to Preserve Fine Ultrastructure of Biological Organizations for Scanning Electron Microscopy, Helium Ion Beam Microscopy and Fluorescence Microscopy

“…In sharp contrast, biological organisms, with lipid-rich membranes encompass cytoplasm containing >80% of water, are extremely challenging objects to apply for SEM. To apply such biological organisms, ethanol dehydration was developed based on the assumption that ethanol was a mild organic solvent that caused less damage than most organic solvents, while concerns over the loss of lipids prompted a search for alternative reagents or methodology 2,3 . The application of osmium tetroxide to fix unsaturated lipids 4 , variable derivatives of organic solvents, or lowering ethanol temperature were introduced to minimize the loss 5 .…”