A Study of Osmium Tetroxide Fixation

Burkl, W; Schiechl, H

doi:10.1177/16.3.157

Cited by 23 publications

(4 citation statements)

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“…10.44). Before our present study, it was established that OsO4 binds to (8) and can partially cross-link some proteins (19.40. 56), but it was also found that OsO4 oxidizes some amino acids (16).…”

Section: Previous Work On the Reaction Of Os04 With Proteinsmentioning

confidence: 87%

Actin filament destruction by osmium tetroxide

Maupin-Szamier¹,

Pollard²

1978

The Journal of Cell Biology

345

130

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We have studied the destruction of purified muscle actin filaments by osmium tetroxide (OsO4) to develop methods to preserve actin filaments during preparation for electron microscopy. Actin filaments are fragmented during exposure to OsO4. This causes the viscosity of solutions of actin filaments to decrease, ultimately to zero, and provides a convenient quantitative assay to analyze the reaction. The rate of filament destruction is determined by the OsO4 concentration, temperature, buffer type and concentration, and pH. Filament destruction is minimized by treatment with a low concentration of OsO4 in sodium phosphate buffer, pH 6.0, at 0~ Under these conditions, the viscosity of actin filament solutions is stable and actin filaments retain their straight, unbranched structure, even after dehydration and embedding. Under more severe conditions, the straight actin filaments are converted into what look like the microfilament networks commonly observed in ceils fixed with OsO4. Destruction of actin filaments can be inhibited by binding tropomyosin to the actin. Cross-linking the actin molecules within a filament with glutaraldehyde does not prevent their destruction by OsO4. The viscosity decrease requires the continued presence of free OsO4. During the time of the viscosity change, OsO4 is reduced and the sulfur-containing amino acids of actin are oxidized, but little of the osmium is bound to the actin. Over a much longer time span, the actin molecules are split into discrete peptides.

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Section: Previous Work On the Reaction Of Os04 With Proteinsmentioning

confidence: 87%

Actin filament destruction by osmium tetroxide

Maupin-Szamier¹,

Pollard²

1978

The Journal of Cell Biology

345

130

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“…Many steps in the classic en bloc electron microscopy staining protocols are based on passive diffusion of chemicals into the tissue. Passive diffusion is one of the main bottlenecks for preparing large samples (smallest dimension >1mm) for electron microscopy (Burkl and Schiechl 1968;Medawar 1941). For small samples (smallest dimension <1mm) a typical en bloc staining protocol takes about 10 -15 days including sectioning (Tapia et al 2012).…”

Section: Introductionmentioning

confidence: 99%

In situ X-ray assisted electron microscopy staining for large biological samples

Ströh

et al. 2021

Preprint

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Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here we present an in situ time-lapsed X-ray assisted staining procedure that opens the "black box" of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in reduced osmium solution. X-ray assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards in silico staining experiments and computational optimization of staining protocols for large samples. Hence, X-ray assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys or humans.

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“…The present study shows that by taking advantage of the knowledge of penetration rates of fixatives (3,4) and by using 2,2-dimethoxypropane as dehydrating agent (5) the processing time can be reduced to three hours without compromising but rather improving the quality.…”

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confidence: 86%

A rapid fixation technique of epidermis for electron microscopy

Andersson

1985

Acta Derm Venereol

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A rapid processing technique for ultrastructural studies of human epidermis has been devised in order to reduce dislocation of soluble compounds and to make available sections for diagnostic purposes within reasonable time (ca 4 hours). The morphology of cellular components agreed with or improved upon that obtained after commonly used methods. Thus, for example, the substance in the intercorneal space was better preserved and the cytomembrane and certain of its specializations appeared more distinct.

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A Study of Osmium Tetroxide Fixation

Cited by 23 publications

References 0 publications

Actin filament destruction by osmium tetroxide

Actin filament destruction by osmium tetroxide

In situ X-ray assisted electron microscopy staining for large biological samples

A rapid fixation technique of epidermis for electron microscopy

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