Fixation of Neural Tissues for Electron Microscopy by Perfusion With Solutions of Osmium Tetroxide

Palay, Sanford L.; McGEE-RUSSELL, S. M.; Gordon, Spencer; Grillo, Mary A.

doi:10.1083/jcb.12.2.385

Cited by 507 publications

(121 citation statements)

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“…In extreme cases the nuclear envelope buckled and the nuclear substance completely aggregated into discrete chromatin blocks. This change was produced in some degree by incorporation of veronal-acetate buffer, as in Palade's fixative: it was more marked after inclusion of isotonic NaCl as well, as in Zetterqvist's fixative; and it was extreme after incorporation of 0-5 per cent CaCl2, as in the fixative of Palay, McGee-Russell, Gordon and Grillo [1962]. Nucleoli were rarely seen in the small lymphocytes.…”

Section: Resultsmentioning

confidence: 87%

“…In preliminary exercises on fixation of fresh rat thymus the above fixative gave better results than the osmic acid formulations of Palade [1952], Caulfield[1957], Zetterqvist [1956] and Dalton [1955], or 1 per cent osmic acid in T9 culture medium. It was superior for three main reasons: preservation of lymphocyte structure, especially the cell membrane and mitochondria, was more complete; fixation was quite uniform throughout the block, and very thin sections (in araldite) were easier to cut.…”

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confidence: 99%

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ULTRASTRUCTURAL CHANGES IN LYMPHOCYTES EXPOSED TO NOXIOUS AGENTS IN VITRO

Trowell

1966

Exp Physiol

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The cytopathological changes produced in lymphocytes by various noxious agents applied in vitro were studied under the electron microscope.Four different lesions were found: (1) the 'anoxic' lesion, produced by anoxia or cyanide, characterized by gross watery swelling of all the cytoplasmic components and internal breakdown of the nucleus; (2) the lesion produced by SH poisons, characterized by swelling of the cytoplasmic matrix and destruction of the cell and nuclear membranes; (3) the 'lytic' lesion, produced by tear gases and chloroform, characterized by rapid onset of cytoplasmic lysis, and (4) the 'radiomimetic' lesion, produced by X-rays, cortisone, mitotic poisons, barbiturates, and glucose deprivation, in which the most striking feature was an increased number of ribosomes.The results suggest that radiation, cortisone, mitotic poisons and barbiturates kill lymphocytes by interfering with the uptake or early metabolism of glucose by the cells.LYMPHOCYTES maintained in vitro, in organ cultures of rat lymph nodes, can be directly killed by low doses of X-radiation [Trowell, 1952] and by low concentrations of cortisone [Trowell, 1953], various mitotic poisons [Trowell, 1960], and barbiturates [Trowell, 1958]. In this respect lymphocytes are more sensitive than any other cells of the body. They are, however, relatively resistant to the toxic action of C02, ammonia, ethanol or urethane, and to rather wide variations in the extracellular levels of H, K, Ca and Cl ions; but their sensitivity to anoxia and metabolic poisons such as cyanide, iodoacetate, arsenic and mercury is similar to that of other cells [Trowell, unpublished]. The reason why lymphocytes are specifically sensitive to the cytocidal action of radiation, cortisone, mitotic poisons and barbiturates is not known, and indeed we have no idea how or why any of these agents damage the cells in the first place.In the above-mentioned experiments the lymphocytes were examined with the light miicroscope, both fresh and after fixation; and, apart from watery swelling of the cytoplasm which was seen inconsistently in a few cases, the earliest morphological change observed was nuclear pyknosis, and the cytological appearances were exactly the same in all cases. The cytoplasm of small lymphocytes is, of course, very tenuous and notoriously difficult to preserve in cytological preparations, so our failure to observe any early or specific signs of cell damage was not altogether unexpected. The more important of these experiments have now been repeated with organ cultures of both lymph nodes and thymus, and the cells have been examined with the electron microscope. As a result I am able to report here some striking differences in the early morphological damage produced by different agents. 207 METHODSOrgan Culture. -The technique of Trowell [1959] was used with the following modifications: the culture grids were made of titanium; Type I culture chambers [Trowell, 1954] were used, and the culture medium was the modification designated T9 [Trowell, 1963]. The orga...

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Section: Resultsmentioning

confidence: 87%

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confidence: 99%

ULTRASTRUCTURAL CHANGES IN LYMPHOCYTES EXPOSED TO NOXIOUS AGENTS IN VITRO

Trowell

1966

Exp Physiol

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“…Fox & Barnard (1957) arrived at figures which correspond to an average of about 3-2 fibres per square micron crossing through the dendrites of the Purkinje cells. Within the parallel fibre bundles the densities will be higher; and rough measurements from published electron micrographs of cross-sections of parallel fibre bundles (Palay et al 1962;Fox et al 1967;Gobel, 1968) (Keynes & Ritchie, 1965) and in the cat sural and hypogastric nerves (Gasser, 1955) give packing densities of 1.5-2*5 fibres per square micron within bundles. Thus the parallel fibres are considerably more densely packed than mammalian peripheral C-fibre bundles.…”

Section: Discussionmentioning

confidence: 99%

An extreme supernormal period in cerebellar parallel fibres

Gardner-Medwin¹

1972

The Journal of Physiology

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SUMMARY1. The electrical responses produced by stimulation at the surface of the cerebellar cortex have been studied in anaesthetized cats.2. The propagation of the underlying activity is attributed to parallel fibres, but the mode of generation of the potential changes is less certain.3. The threshold for a second response is up to 40 % lower following a conditioning stimulus, and the responses to test stimuli less strong than the conditioning stimulus are correspondingly potentiated.4. A second stimulus given 22 msec after the first produces a response which propagates 15-20 % faster along the folium.5. The reduction of the threshold and the increase of the propagation velocity are as large with small (but above threshold) conditioning shocks as with large shocks.6. Both after-effects are present from 5 to 100 msec after a conditioning stimulus, with maximal values at about 10-30 msec.7. The fibre conduction velocity inferred from collision experiments agrees with that inferred from the propagation velocity of the responses, and shows a similar increase after conditioning.8. A second shock does not recruit a new and faster population of parallel fibres.9. Under the conditions of electrical stimulation the after-effects are probably restricted to those fibres which are active during conditioning.

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“…The goal of fixation is to preserve cells and tissue constituents in as close a life-like state as possible by preventing postmortem decay. However, the dilemma of fixation has always been that it introduces some artifact, which is observed as alterations of the original chemical and physical compositions of tissues 14,15 . A proper fixation protocol is crucial, as well for the studies on histomorphometric evaluation of peripheral nerve fibers particularly concerning the quantitative features such as axon number, axonal cross-sectional area and 2,4 .…”

Section: Discussionmentioning

confidence: 99%

“…A proper fixation protocol is crucial, as well for the studies on histomorphometric evaluation of peripheral nerve fibers particularly concerning the quantitative features such as axon number, axonal cross-sectional area and 2,4 . Most commonly used fixation protocols in these studies are immersion and perfusion fixation techniques 5,14,16 . Although transcardial perfusion has been approved as the most effective method it indeed has several disadvantages.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Fixation Methods for Peripheral Nerve Fiber

Ulkay¹,

Aktaş²,

Aksoy³

et al. 2013

Kafkas Univ Vet Fak Derg

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Accurate fixation is a must for the assessment of myelinated nerve fiber morphology and transcardial perfusion and immersion methods are the most commonly used fixation techniques. In the present study we designed a new fixation technique for the histomorphometric and stereological evaluation of sciatic nerve fiber and referred it as instillation fixation. The method involved preliminary in situ fixation of the nerve sample without dissecting it from the animal body followed by a complete conventional fixation protocol. The objective of this study was to compare the three fixation techniques with each other for fixation artifacts. Eighteen female Wistar albino rats constituted the study material. The animals were allocated into three experimental groups corresponding to different fixation methods (immersion, instillation and transcardial perfusion, respectively). Quantitative assessments of nerve samples harvested from the animals of each group included the number of total myelinated axons, normal myelinated axons, alterations in myelin compaction, myelinated axons with irregular fiber shape, and with myelin loops and g ratio. Results revealed that normal myelinated axons were markedly lower in the immersion fixation group compared to those of instillation and transcardial perfusion. Moreover a significant decrease was noted with respect to alterations in myelin compaction in the instillation fixation group. In contrast, no significant difference was observed in myelin thickness and axon cross sectional area. In conclusion instillation fixation technique proved to be a valid and simple method for the assessment of peripheral nerve morphology for further analyses in a rat model. Keywords: Rat, Sciatic nerve, Fixation method Periferal Sinir Tespit Yöntemlerinin KarşılaştırılmasıÖzet Doğru bir tespit işlemi miyelinli periferik sinirlerin morfolojik değerlendirmesi için ön koşuldur. Kalp perfüzyonu ve daldırma en yaygın kullanılan tespit yöntemlerindendir. Çalışmamızda bu metotlara ek olarak damlatma tekniği olarak adlandırılan yeni bir tespit metodu uygulandı. Sinir doku örneği canlı hayvan üzerinden çıkarılmadan ön tespit işlemine tabi tutulduktan sonra rutin tespit işlemleri tamamlandı ve üç tespit yönteminin tespit artefaktları açısından birbirleriyle histomorfometrik ve stereolojik olarak karşılaştırılması gerçekleştirildi. On sekiz adet dişi Wistar albino sıçan kullanıldı. Hayvanlar sırasıyla daldırma, damlatma ve kalp perfüzyonu olmak üzere üç eşit gruba ayrıldı. Her bir gruptaki hayvanlara ait siyatik sinir örneklerinin kantitatif değerlendirilmesi toplam miyelinli akson sayısı, normal miyelinli akson sayısı, miyelin kompaksiyonundaki değişimler içeren aksonların sayısı, düzensiz demet yapısı ile kıvrımlı miyelin halkaları içeren miyelinli aksonların sayısı ve g oranları hesaplanarak gerçekleştirildi. Elde edilen bulgulara göre daldırma tespit grubundaki normal miyelinli akson sayısında, damlatma ve kalp perfüzyonu grupları ile karşılaştırıldığında belirgin bir düşüş belirlendi. Bununla beraber damlatma tesp...

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Fixation of Neural Tissues for Electron Microscopy by Perfusion With Solutions of Osmium Tetroxide

Cited by 507 publications

References 24 publications

ULTRASTRUCTURAL CHANGES IN LYMPHOCYTES EXPOSED TO NOXIOUS AGENTS IN VITRO

ULTRASTRUCTURAL CHANGES IN LYMPHOCYTES EXPOSED TO NOXIOUS AGENTS IN VITRO

An extreme supernormal period in cerebellar parallel fibres

Comparison of Fixation Methods for Peripheral Nerve Fiber

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