Lipid losses during processing of cardiac muscle for electron microscopy

Abstract

SUMMARY The lipid retaining properties of several methods of processing tissue for electron microscopy (EM) have been assessed quantitatively. Guinea‐pig hearts were perfused in vitro at 37°C with 3H‐oleic acid bound to albumin. The hearts were fixed by perfusion with 4% glutaraldehyde in 0.1 M cacodylate buffer. Pieces of left ventricle and interventricular septum were removed., weighed and processed for EM. The fluids used at each stage of processing were monitored for loss of radioactive lipid by scintillation counting. Lipids were extracted from the processed tissue immediately before the embedding stage using a mixture of chloroform: methanol (2:1 v/v). Counts from processed tissue were compared with counts from tissue extracted directly after perfusion fixation in order to monitor subsequent losses during processing. A modified version of Epon processing, omitting 100% ethanol, acetone or propylene oxide, gave a lipid retention of only 20.6%. The addition of para‐phenylenediamine to the procedure did not improve the retention although this has been shown to be a useful stain for intracellular lipid. Water soluble Durcupan which does not involve ethanol or acetone dehydration has an average retention of 63% with 100% recovery while the lesser known polymer GACH, a mixture of glutaraldehyde and carbohydrazide used both for dehydration and embedding showed a lipid retention of 82% of the counts recovered although recovery was only 69%. An attempt was made to determine which classes of lipids were present in the tissue after perfusion fixation using thin layer chromatography. It was found that the presence of any of the processing fluids affected the polarity of the lipids and their rates of migration on thin layer plates.

Contribution of osmium tetroxide to the image quality and detectability of iron in cells studied by electron spectroscopic imaging and electron energy loss spectroscopy

“…They also found that a mixture of glutaraldehyde and OsO, as a fixative was superior to glutaraldehyde followed by OsO, in retaining lipids. An earlier study using i3H1 oleic acid (Ward and Gloster, 1976) showed the largest loss (33.7%) was found in the dehydration phase of liquid fixation of the myocardium tissue samples.…”

“…A number of previous studies have used this approach in relationship to a number of chemical constituents of cells. Lipid soluble constituents were lost predominantly in the ethanol dehydration phase (Mizuhira et al, 1981;Ward and Gloster, 1976). Mizuhira et al (1981) showed that 3H-cholesterol loss was usually limited to ethanol dehydration.…”

Radioautographic Study of the Incorporation of (3H)-Choline Into the Phospholipids of Secretory Ameloblasts and Enamel of Normal and Essential-Fatty-Acid-Deficient Rats

“…The extraction is generally considered to be unspecific, since it does not discriminate between the various classes of lipids. although all the phosphatidylcholine and only half of the phosphatidylethanolamine are lost in the rat liver (Morgan and Huber, 1967;Williams, 1968, 1969a,b;Ward and Gloster, 1976;Weibull et al, 1983).…”

Glutaraldehyde: A review of its fixative effects on nucleic acids, proteins, lipids, and carbohydrates

“…These investigators found that perfusion fixation with GA did not cause significant changes in the levels of the brain lipid classes that they measured, including total lipids, cholesterol, or phosphatides. While not a pure GA fixation study, another study used radiolabeling to measure lipid extraction following combination GA and carbohydrazide perfusion fixation in cardiac tissue (Ward & Gloster, 1976). These investigators found that their combination GA and carbohydrazide fixation method allowed for the retention of 82% of lipids following chloroform-methanol treatment.…”