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At least 19 recordsLinked to original sources
PubMed1993

Observation of 100 nm periodic fibrils in mouse corneal stroma by cryoultramicrotomy.

We observed mouse corneal stroma after treatment with 20 mM adenosine 5'-triphosphate (ATP), thin-sectioned by cryoultramicrotomy, with a transmission electron microscope. A wide array of 100 nm periodic fibrils (100 nm PFs) was observed among D-periodic collagen fibrils. By indirect immuno-cryoultramicrotomy with anti-type VI collagen antibody, immunogold particles were located on 100 nm PFs, indicating that 100 nm PFs were composed of type VI collagen. Cryoultramicrotomy is useful for studying the ultrastructure of the extracellular matrix of the corneal stroma.

Adenosine Triphosphate↗
PubMed1983

Cryoultramicrotomy versus plastic embedding: comparative immunocytochemistry of rat anterior pituitary cells.

The anterior pituitary of the rat is used as a model for the study of the effects of freezing or plastic embedding on the maintenance of antigenicity. Rat anterior pituitaries are fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4. Some of the blocks are post-fixed before being divided into two lots. One batch is frozen, while the other is dehydrated and embedded. The indirect antibody enzyme method is applied to ultrathin sections obtained by cryoultramicrotomy after freezing or by sectioning after embedding. All six pituitary hormones are detected by both methods. Comparison shows that the morphological characteristics are identical for both techniques, though ultrastructural preservation is better after embedding. Immunoreactivity is found in secretory granules and sometimes in the endoplasmic reticulum. Osmium postfixation may reduce or even abolish antigenicity in plastic-embedded tissue. After cryoultramicrotomy, however, even after osmium fixation, antibody may be used 1000 times more diluted than after plastic embedding. Embedding preserves ultrastructure and limited antigenicity while the use of cryoultramicrotomy is a far more sensitive technique.

Adrenocorticotropic Hormone↗
PubMed2005

Application of cryofixation and cryoultramicrotomy for biological electron microscopy.

Conventional chemical fixation and embedding of specimens in resins are accompanied by many artifacts, including postmortem structural alterations. Antigenicity of constituents of specimens can be deteriorated and soluble elements relocated in the process of chemical fixation and resin-embedding. Cryofixation and cryoultramicrotomy will overcome many of these drawbacks of chemical fixation and resin embedding. The theoretical background, equipment, methods, and applications of cryofixation and cryoultramicrotomy for biological specimens are reviewed.

Cryopreservation↗
PubMed1991

Immunogold labelling in combination with cryoultramicrotomy, freeze-etching, and label-fracture.

During the past years, the methods of ultrastructural visualization of intracellular and cell-surface proteins have been improved considerably, mainly as the result of the development of low-temperature preservation in combination with immunocytochemical labelling procedures using poly- or monoclonal antibodies. In this contribution we will discuss the combination of immunogold labelling with cryoultramicrotomy and two replica methods, i.e. freeze-etching and label-fracture. The main advantage of cryoultramicrotomy is that it enables post-sectioning labelling, thus providing complete accessibility of all cellular antigens, located both intracellularly and on the cell surface. Important parameters that influence the labelling (i.e. label-efficiency), including penetration of the label and antibodies in the section, effects of fixatives on antigenicity, and steric hindrance, will be discussed in detail. The replica methods have the advantage of enabling an analysis of the lateral distribution of antigens located at the cell surface. The label efficiency is of particular importance in these studies and in this context several parameters will be discussed, including accessibility and effect of fixatives.

Animals↗
PubMed1989

Cryoultramicrotomy of ultra-rapidly frozen specimens.

The physical events of the cryoultramicrotomy at the level of organelles and macromolecules are not completely understood. The extent to which tissue is either cut by the edge of the knife or fractured ahead of the knife is one such event. This issue of cryofracturing versus cryosectioning during cryoultramicrotomy has been examined in quick frozen, uncryoprotected rat liver. Cryosectioned specimens were freeze-substituted and edge-on views of the sectioned surface were examined in TEM. In tissue regions showing no obvious ice crystals, fracturing was rare. Regions with less adequate freezing however had numerous fractured structures. These results indicate that high quality freezing promotes sectioning over fracturing and thus works to eliminate this serious artifact.

Animals↗
PubMed1991

Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections.

Ultrathin sections of rapidly frozen, briefly pre-treated muscle tissue are cut and thereafter are thawed and contrasted using a negative staining technique. The method has provided micrographs in which the in-vivo order in the muscle fibres has been preserved well enough to enable both a more complete interpretation of X-ray diffraction evidence from muscle, and also a gain of new ultrastructural information on aspects of myofibril and myofilament architecture in different types of fibre. Examples here are taken from chicken, rabbit and fish muscles and show both the M-band and the bridge region of the A-band in great detail. To enhance the detail in the original images, one-dimensional (1-D) and 2-D averaging techniques (lateral smearing and step averaging, respectively) are used. Although there is major shrinkage in section thickness to about one-third of its original value, demonstrated here for the first time is the fact that the characteristic A-band lattice planes are preserved in these sections in 3-D. This confirms the usefulness of cryosections not just for 1-D and 2-D image processing, but also for 3-D reconstruction. Thus, in combination with techniques of image processing, cryoultramicrotomy can give the muscle morphologist the detailed data that are needed to match the molecular biologists, biochemists and immunologists in the interpretation of their data about physiological and pathophysiological events in muscle fibres at the macromolecular level.

Animals↗
PubMed1992

Cryoultramicrotomy: electrostatic transfer of dry ultrathin frozen sections on grids applied to the central nervous system.

Due to the extreme fragility of ultrathin frozen sections of brain tissue, the cryoultramicrotomy of non-embedded tissue has not been sufficiently used for immunocytochemical studies of the central nervous system. Sections are easily disrupted by the liquid surface tension when a droplet of sucrose is used (method by Tokuyasu, 1973) for their transfer on the grids. Use of silicotungstic acid (Tsuji, 1986) in place of sucrose improved the preservation of the ultrastructure but still could not resolve the difficulty. This report describes a new procedure for transferring dry ultrathin frozen sections by means of electrostatic attraction induced on the membrane covering the grids. Once attached electrostatically to the membrane, the sections were retained by van der Waals' forces. The dry ultrathin frozen sections obtained from both fresh and fixed brains displayed good preservation of their ultrastructures over a large surface. This new method which electrostatically transfers dry ultrathin frozen sections, avoiding the use of any liquid, is expected to serve for the immunocytochemical identification of neuronal cell bodies and terminals as well as their neurotransmitters and enzymes in both fresh and fixed brains.

Animals↗
PubMed1991

[Principles and application possibilities of cryoultramicrotomy].

This paper reviews the technical aspects of cryoultramicrotomy. The key preparation steps in the technique, namely freezing, sectioning, thawing, and staining are analysed. The application of this method to immunocytochemistry and analytical electron microscopy are discussed with respect to the present possibilities and problems.

Cryoultramicrotomy↗
PubMed1991

Cellular location of thymus-leukemia (TL) antigen as shown by immuno-cryoultramicrotomy.

Thymus-leukemia (TL) antigen is a class I molecule of the major histocompatibility complex that is expressed on the surface of mouse cortical thymocytes. Though not expected, it has been reported that TL antigen can be found on isolated mitochondria of TL+ cells. We used immuno-cryoultramicrotomy to look for TL on mitochondria in situ, thereby avoiding the plasma membrane contamination that occurs when isolating organelles. Establishing optimal fixation conditions was crucial, as mitochondrial structure was not preserved by the low concentrations of fixative needed for detection of antibody labeling. The plasma membranes of tissue culture and thymus cells were labeled well with anti-TL antibody and protein A-gold conjugate, while mitochondria within the cells were not labeled. Isolation of mitochondria on a one-step Ficoll gradient resulted in a purer organelle preparation than did isolation of mitochondria by centrifugation alone. Generally, mitchondria within this purer preparation were not labeled. Our data show that under conditions where contamination by plasma membrane is not a major concern, TL antigen cannot be detected on mitochondria.

Animals↗
PubMed1991

Presence of renin, angiotensinogen, angiotensin II in the lamb anterior pituitary gland: immunocytochemical study after cryoultramicrotomy.

The presence of renin, angiotensin I-converting enzyme and angiotensin II detected by immunocytochemistry in the adult male rat anterior pituitary has suggested the existence of a pituitary renin-angiotensin system. To establish another mammalian experimental model we have investigated the presence of renin, angiotensinogen, angiotensin I-converting enzyme, and angiotensin II II in five normal lamb anterior pituitaries by immunocytochemistry after cryoultramicrotomy. Renin, angiotensinogen and angiotensin II immunoreactivities were observed only in cytoplasmic granules of lactotrophs, and the three proteins were found co-localized with prolactin in the same granules by double immunolabelling. No immunoreactive angiotensin I-converting enzyme was observed. These results suggest an activation of renin in the cytoplasmic granules of lactotrophs leading to a local synthesis of angiotensin II. Thus, the lamb anterior pituitary may provide a good experimental model for investigating the possible autocrine action of a local renin-angiotensin system on prolactin release in the human pituitary.

Angiotensin II↗
PubMed1979

Electron probe X-ray microanalysis and cryoultramicrotomy of unstained myocardial sarcoplasmic reticulum, in situ and fragmented.

Myocardial sarcoplasmic reticulum of cats in situ and fragmented sarcoplasmic reticulum (FSR) were analysed using X-ray microanalysis, cryoultramicrotomy and scanning transmission electron microscopy. 2 types of FSR vesicles can be distinguished morphologically and by their different elemental composition especially by different Ca loading. The Ca content of the sarcoplasmic reticulum can also be detected in situ.

Animals↗
PubMed1985

Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique.

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.

Breast↗
PubMed1989

Presence of angiotensin II in the adult male rat anterior pituitary gland: immunocytochemical study after cryoultramicrotomy.

Angiotensin II (AII)-like immunoreactivity and binding sites have recently been demonstrated at the pituitary level. This peptide also exerts a stimulatory effect on anterior pituitary hormone release. Immunocytochemistry on ultrathin sections obtained by cryoultramicrotomy was used with the aim of localizing endogenous AII-like material at the cellular and subcellular levels of the anterior pituitary gland. AII-like immunostaining was observed only in gonadotrophs, lactotrophs, and corticotrophs. In gonadotrophs, AII-like immunoreactivity was restricted only to secretion granules. In the two other immunoreactive cells, lactotrophs and corticotrophs, immunostaining was observed in the cytoplasm and in the nucleus. In the cytoplasm, AII-like material was visualized in the cytoplasmic matrix and in the secretory granules. In the nucleus, immunostaining was distributed in the euchromatin in the vicinity of the heterochromatin. AII-like immunoreactivity was also seen at the plasma membrane, but only scarcely. No reaction product was found when anti-AII serum preincubated with AII was used. These immunocytochemical results (1) provide evidence that gonadotrophs are only a site of synthesis and/or storage of AII-like material, (2) indicate that lactotrophs and corticotrophs are cells for AII and (3) provide cytological evidence for a direct participation of AII in the regulation of the lactotropic and corticotropic function.

Angiotensin II↗
PubMed1975

Ribonucleoprotein components in liver cell nuclei as visualized by cryoultramicrotomy.

The interphase nucleus of the normal rat hepatocyte has been studied in ultrathin frozen sections after glutaraldehyde fixation and the modification of various staining procedures known to be specific for DNA structures (Moyne's thallium stain, Gautier's osmium-ammine) or preferential for RNP carriers and basic proteins (regressive stains based on the use of EDTA or citrate, negatively charged colloidal iron). The results are comparable to those obtained after classical dehydration and embedding. Particular attention has been paid to the nucleolus and extranucleolar RNP components, such as perichromatin fibrils and granules, as well as interchromatin granules. A striking observation was the uneven size and the strongly increased number of perichromatin granules, and the appearance of a contiguous interchromatin net, containing nucleoproteins. Cryoultramicrotomy without embedding appears to be very useful for the exploration of the nucleus in thick sections which remain sufficiently transparent even with the usual accelerating voltages.

Animals↗
PubMed1982

Imaging of outer segment periodicities in unstained cryoultramicrotomy sections of the frog retina.

Frog retinas were examined following cryoultramicrotomy. The extent of glutaraldehyde cross-linking ranged from maximally 2% for 2 h to no cross-linking. When the tissue was cryoprotected, the effects of hypotonic, isotonic and hypertonic media on outer segment banding periodicities were studied. The specimen was cryosectioned at 193 K to 113 K with sections collected on a dry knife (213 K to 123 K). The sections were freeze-dried and only rehydrated with phosphotungstic acid section stain when transmission electron microscopy was used to establish the extent of gross retinal morphology for each fixation protocol employed. As the extreme of no cross-linking and no cryoprotection was approached and reached, conventional sections which displayed extensive retinal morphology were not obtained. Instead, fragments of retinal tissue resulted which showed periodicities of alternating light and dark bands. These periodicities are interpreted as photoreceptor outer segment discs with contrast formation depending upon an interplay between three factors: mass density differences between disc and interdisc space, section cutting angle, and section thickness.

Animals↗
PubMed1986

Application of cryoultramicrotomy to immunocytochemistry.

This paper reviews the most recent status of immuno-cryoultramicrotomy. The technical aspects of each step of the method are also analysed in detail with the intention of providing a useful source of information for investigators using this method.

Animals↗
PubMed1983

Ultrastructural aspects after cryoultramicrotomy of bone tissue and sutural cartilage in neonatal mice calvaria.

Using transmission electron microscopy after cryoultramicrotomy, mineralized as well as unmineralized bone tissues and sutural cartilage were observed in neonatal mice calvaria. A good definition of osteoblasts (nucleus, membranes, organelles) and extracellular constituents (collagen fibrils, matrix vesicles, mineral substance) was obtained. The sutural zone was composed of an unmineralized cartilaginous tissue with more or less hypertrophic cells surrounded by a finely fibrillar matrix.

Animals↗
PubMedDate not supplied

Cryoultramicrotomy as a preparative method for x-ray microanalysis in pathology.

The role of ion shifts in cell injury has become one of the most intriguing fields of application of X-ray microanalysis in pathology. In principle, cryo(ultra)-microtomy is the best preparative method for X-ray microanalysis of diffusible substances. In this review, the following points are discussed: the choice of freezing method and coolant, the use of high molecular weight polymer cryoprotectants and their possible physiological effects the choice of the cryosectioning temperature, and techniques for handling and transfer of sections. Experiences with the sectioning of cryoprotected tissue are presented. Finally, other preparative techniques for microanalysis are compared to cryoultramicrotomy.

Animals↗