The Osmotic Effects of Electron Microscope Fixatives

Bone, Q.; Denton, E. J.

doi:10.1083/jcb.49.3.571

Cited by 165 publications

(80 citation statements)

References 15 publications

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“…Bone & Denton (1971) found that a hypotonic buffer rinse of osmolality equivalent to 60 % of that of the body tissue fluid produced the least volume change in isolated fish scales. Osmium tetroxide, on the other hand, destroys the osmotic properties of membranes, and the osmolality of the buffer is then relatively unimportant.…”

Section: Distortion Due To Histological Fixationmentioning

confidence: 93%

Quantitative study of the non‐circularity of myelinated peripheral nerve fibres in the cat

Arbuthnott

Ballard

Boyd

et al. 1980

The Journal of Physiology

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SUMMARY1. One hind limb of each of four cats was either chronically de-efferentated, or chronically de-afferentated, and perfused with buffered glutaraldehyde fixative. Up to three different muscle nerves were dissected from each limb, post-fixed in osmium tetroxide and embedded in Epon. Ultrathin transverse sections were mounted on Formvar-coated single-hole specimen grids so that all the fibres in each nerve could be examined individually by electron microscopy.2. Non-circularity was expressed as the ratio (0): axon cross-sectional area area of a circle with same perimeter'The degree of non-circularity of all the afferent axons, or all the efferent axons, in each muscle nerve was determined. The proportion of fibres cut through the paranodal region, or through the Schwann cell nucleus, was as expected for group I afferent and for a and y efferent fibres, but hardly any typical paranodal sections of group II or III afferent fibres were encountered which suggests that their paranodal arrangement differs from that of other groups. In a quantitative comparison of noncircularity in different functional groups, fibres cut through paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were rejected.3. All the y efferent fibres in one nerve were studied in a series of sections cut at 25 ,tm intervals. The degree of non-circularity was found to be relatively constant along the internode of most fibres when the values at paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were ignored.4. The value of 0 varied widely from 1-0 (circular) to 0-5 or less from fibre to fibre within every functional group. However, the mean value of qS was less for y axons (0.68) than for a axons (0.78), and less for group III axons (0.79) than for axons in groups I and II (both 0.84). When the results for all the nerves were aggregated, these differences were statistically very highly significant, as was the difference in qS between group I and a fibres. If values of qS < 0 5 were rejected, the difference between the between a and y fibres was still very highly significant. 5. The external perimeter (S) of a non-circular fibre differs from 2i times the diameter of a circle just enclosing the fibre (D). It is shown that S = 0 95 7TD for group I and II fibres, S = 0 90 nrD for a and group III fibres, and S = 0-85 iD for y fibres.6. The myelin period, or interperiod repeat distance, varied from 14 1 to 15-6 nm in different cats, implying radial shrinkage of the myelin sheath from 15 to 23% The myelin period in a particular cat was the same for several nerves, and the same for fibres in different functional groups.7. The possibility that repetitive firing of axons during fixation contributed to the varying degree of non-circularity is considered but rejected as unlikely.8. It is deduced that about 10 % radial shrinkage of the myelin sheath, but little or no osmotic shrinkage of the axon, occurred during fixation and rinsing. Further radial shrinkage of about 8 % in all components of the fibre probably occurred as a result of subsequen...

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Section: Distortion Due To Histological Fixationmentioning

confidence: 93%

Quantitative study of the non‐circularity of myelinated peripheral nerve fibres in the cat

Arbuthnott

Ballard

Boyd

et al. 1980

The Journal of Physiology

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“…The sample was then fixed for 1-25 h in 2 % glutaraldehyde, buffered with 100 mM-cacodylate. This is a buffer osmolarity -60% that of normal tissue fluids, as recommended by Bone & Denton (1971) for optimum preservation of cell volume in the face of the reduction of cytoplasmic osmolarity caused by cross-linking fixatives. The same buffer was used for the subsequent wash, and as the vehicle for 1 % osmium tetroxide secondary fixation.…”

Section: Electron Microscopical Stereologymentioning

confidence: 99%

A phosphorus nuclear magnetic resonance study of metabolites and intracellular pH in rabbit vascular smooth muscle.

Spurway

Wray

1987

The Journal of Physiology

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SUMMARY1. 31P nuclear magnetic resonance (n.m.r.) spectroscopy was used to investigate metabolites, intracellular pH (pHi) and the effects of pHi on tone in rabbit blood vessels. The vessels were bathed in mammalian Ringer solution and maintained at 20 'C while inside the spectrometer.2. Vascular spectra showed relatively low phosphocreatine (PCr) concentrations compared to skeletal muscle. The [PCr]/[ATP] ratio was only 1-32 + 0-09 (n = 7). There was also a prominent phosphomonoester (PME) peak. Similar features have been reported for other smooth muscles examined by 31P n.m.r. 5. The effects of NH4Cl upon pH were investigated in metabolically inhibited preparations. During 10 min applications of 30 mM-NH4Cl (isosmotically substituted for NaCl) the pHi rose; during subsequent NH4Cl removal it fell below control values. In the least inhibited tissues the total pHi excursion between NH4Cl applications and removals was 0 5 unit.6. Rabbit ear vessels have been found to increase vascular tone during manoeuvres which were expected to decrease pHi. From the direct measurement of pHi reported in this study, it is concluded that the vascular tone changes brought about by NH4Cl application and withdrawal may be attributed to the alteration of pHi.

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“…Careful fixation with aldehyde mixtures does not generally degrade cell plasma membranes sufficiently to admit antibody-sized proteins (33,34). The experiment of Fig.…”

Section: Surface Localization Of Trk2p Via the C-terminal Egfpmentioning

confidence: 99%

Epitope Tagging of the Yeast K+ Carrier Trk2p Demonstrates Folding That Is Consistent with a Channel-like Structure

Zeng

Pypaert

Slayman

2004

Journal of Biological Chemistry

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The Osmotic Effects of Electron Microscope Fixatives

Cited by 165 publications

References 15 publications

Quantitative study of the non‐circularity of myelinated peripheral nerve fibres in the cat

Quantitative study of the non‐circularity of myelinated peripheral nerve fibres in the cat

A phosphorus nuclear magnetic resonance study of metabolites and intracellular pH in rabbit vascular smooth muscle.

Epitope Tagging of the Yeast K+ Carrier Trk2p Demonstrates Folding That Is Consistent with a Channel-like Structure

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