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PubMed1981

Further light microscopic studies on morphology and development of Pneumocystis carinii.

In order to add more advance in light microscopic investigation of P. carinii, phase contrast microscopy partly followed by wet giemsa stain and semiultrathin section of the lungs embedded in JB-4 plastic were studied. In phase contrast microscopy, small and large sized trophozoites of P. carinii were clearly recognized. Although movement of trophozoite was not found, rhythmic movement of intracystic bodies with filopodia was often seen in mature cyst. Those living organisms were then directly stained with Giemsa by infiltrating under the coverglass. Thus the organism could be investigated both in unstained and stained conditions. It is noticed with interest that 8 intracystic bodies seem to fill up the cavity of cyst when cell division is completed, then they liberate and become independent into spherical bodies, followed by banana-shaped or amoeboid forms with motility. An emphasis was done that semiultrathin section made from JB-4 plastic embedded lungs was quite useful for investigation of P. carinii infection. Several sizes of mononuclear thin-walked trophozoites, mature and immature cysts, and empty cysts were more clearly distinguished than any other light microscopical method ever reported.

Animals↗
PubMed1982

Low temperature techniques applied for CTEM and STEM analysis of cellular components at a molecular level.

One of the most important problems in tissue preparation for electron microscopic analysis at a molecular level involves the preservation of the tissue without introducing extensive denaturation of the proteins. Low temperature is a most efficient condition for the inhibition of protein denaturation and freeze-drying offers favourable conditions for transferring proteins to a dry state with minimal denaturation of the proteins. However, the embedding of the dried tissue in a plastic leads to extensive denaturation of the proteins when performed in the conventional way. This eliminates very efficiently the advantages of the method. The situation becomes even worse when subjecting the tissue to freeze-substitution. To eliminate as far as possible the denaturing effect of plastic embedding, freeze-drying can be combined with low temperature embedding in a plastic. Freeze-fracturing allows a most efficient use of low temperature to reduce conformation changes in proteins. The value of the freeze-fracturing technique depends entirely on a precise knowledge of the location of the fracture planes. Since this location is not known, it must be determined on the basis of a deduction. If this deduction is wrong, the method becomes misleading. Two methods which allow a certain testing of the correctness of the deduced location of the fracture planes are mentioned.

Animals↗
PubMed1983

Staining plastic sections: a review of problems, explanations and possible solutions.

Many of the difficulties of staining plastic embedded tissues for light and electron microscopy derive from physical exclusion of hydrophilic staining reagents by hydrophobic embedding media. Structures which stain most intensely with hydrophilic reagents usually contain less hydrophobic plastic than do non-staining structures. Such incomplete infiltration is apparently caused by exclusion of viscous, hydrophobic monomers by physically dense and/or well hydrated tissue elements. In keeping with this, generalized staining of tissues embedded in hydrophobic media does occur when hydrophobic reagents are used. Staining of plastic-free structures with single hydrophilic reagents or with sequences of such reagents, is, however, largely rate-controlled. The surprising similarity of hydrophilic and hydrophobic plastic embedding media is discussed. Limits of this simple model are explored, with a consideration of the roles of fixative and of monomer-tissue reactions.

Histological Techniques↗
PubMed1985

A removable polar embedding medium for light microscopy.

A new plastic embedding medium for light microscopy is described. The monomer mixture consists of equal proportions by volume of acrylonitrile, dimethyl acrylamide and methyl methacrylate, and may be polymerized by exposure to ultraviolet light in the presence of benzoin methyl ether as catalyst. Dithiothreitol may also be added to the monomer mix to limit the degree of polymerization. The resulting polymer is soluble in dimethyl formamide.

Acrylonitrile↗
PubMed1994

A simplified technique for histologic analysis of central nervous system tissues using glycol-methacrylate plastic coupled with pre-embedding immunocytochemistry.

Paraffin and some plastic embedding techniques will destroy many antigens routinely detected by immunocytochemistry performed on frozen tissue sections. However, morphologic quality is compromised to varying extents in frozen tissue, even with the use of cryoprotection. We report a simple glycol-methacrylate (GMA) embedding technique using vibratome-sectioned mouse brain reacted for tyrosine hydroxylase (TH) immunoreactivity before plastic embedding. In this study we used a short (4 h) simple, GMA embedding procedure which subsequently provided 1.5-5.0 microns sections yielding morphologic details superior to frozen or paraffin sections. Prior to embedding we used a peroxidase-antiperoxidase (PAP) reaction with the 3,3'diaminobenzidine tetrahydrochloride (DAB) chromogen visualizing TH. Several different counterstains were used, demonstrating the versatility of this embedding procedure.

3,3'-Diaminobenzidine↗
PubMed2000

Immunohistochemical myofiber typing and high-resolution myofibrillar lesion detection in LR white embedded muscle.

We have developed a method of fixing, embedding, sectioning, and staining that allows high-resolution detection of myofibrillar structure and myosin immunocytochemical muscle fiber typing in serial semithin sections of LR White plastic embedded muscle at the light microscopic level. Traditional approaches, such as cryostat sections, permit fiber typing, but small myofibrillar lesions (1-3 sarcomeres) are difficult to detect because of section thickness. Semithin sections of hydrophobic resins do not stain well either histochemically or immunocytochemically. Electron microscopy can resolve lesions and discriminate fiber types based on morphology, but the sampling area is small. Our goal was to develop a rapid method for defining both fiber type and high-resolution primary myofibrillar lesion damage. Mild fixation (1-4% paraformaldehyde, 0. 05-0.1% glutaraldehyde) and embedment in a hydrophilic resin (LR White) were used. Myofibrillar structure was extremely well preserved at the light microscopic (LM) level, and lesions could be readily resolved in Toluidine blue stained 500-nm sections. Fiber type was defined by LM immunomyosin staining of serial plastic semithin sections, which demonstrated reciprocal staining patterns for "fast (Sigma M4276) and "total" (skeletal muscle) myosins (Sigma M7523).

Acrylic Resins↗
PubMed1987

Similarities and variations among lobular carcinoma cells.

A cytomorphological description of lobular carcinoma of the breast is given based on imprints, paraffin-embedded 5-mu sections, and plastic-embedded 2-mu sections in nine patients. Both nuclear and cytoplasmic features are illustrated and tabulated. A basic lobular carcinoma cell is depicted. Cytoplasmic vacuolization is demonstrated in all tumors. Transitions from one lobular cell variation to another are demonstrated. It is suggested that more precise knowledge of the similarities and the variations in the morphology of the lobular carcinoma cell will help in both cytological and histological diagnosis.

Breast Neoplasms↗
PubMed1997

Multifocal brain damage induced by phencyclidine is augmented by pilocarpine.

Phencyclidine and other antagonists of the N-methyl-D-aspartate subtype of glutamate receptor cause psychosis in humans. In low doses these agents induce a reversible neurotoxic reaction in the rat brain that is limited to the retrosplenial granular cortex. Some investigators have reported that phencyclidine at higher doses or by more prolonged treatment causes a more disseminated pattern of damage. However, it has not been clearly demonstrated whether the disseminated damage is reversible or irreversible and whether it is consistently reproducible, nor is it known how many and which neurons are at risk. In the present study we addressed these questions using several histological approaches (plastic-embedded thin sections for light microscopy and ultrathin plastic sections for electron microscopy, paraffin-embedded haematoxylin and eosin sections, 72 kDa heat shock protein immunocytochemistry and de Olmos silver impregnation) to study the lesions induced in rat brain by phencyclidine (alone or when augmented with pilocarpine). We found that phencyclidine can kill a relatively large number of neurons distributed over many cerebrocortical and limbic brain regions, but the multifocal pattern of damage occurred in only a small percentage of treated rats. The addition of a low dose of pilocarpine to phencyclidine caused the widespread pattern of damage to manifest on a much more consistent basis. Available evidence suggests that disinhibition of multiple converging excitatory pathways is the mechanism by which phencyclidine triggers widespread neuronal degeneration; however, the specific combination of excitatory inputs that contributes to the pathological process may differ from region to region.

Animals↗
PubMed1999

A study of the gonadotropin releasing hormone neuronal network in the median eminence of the rhesus monkey ( Macaca mulatta) using a post-embedding immunolabelling procedure.

The purpose of this study was to describe the ultrastructural features of gonadotropin releasing hormone (GnRH) axonal processes in the median eminence of the monkey, using a post-embedding immunogold labelling procedure. Evidence was also sought to evaluate the view that release of this peptide may be governed by direct inputs to GnRH axons in the median eminence. Plastic embedding was used to preserve ultrastructure, and a polyclonal rabbit anti-GnRH was used as primary antibody. Immunogold labelling with 15-nm particles was almost exclusively found overlying dense core vesicles (dcvs) and preabsorption of the primary antibody with synthetic GnRH eliminated this labelling. Morphometric analysis was performed on tissue from two monkeys. Four types of profiles containing GnRH immunoactive dcvs were observed. Type I profiles were morphologically unremarkable with a cross sectional area of approximately 0.6 microm2 and probably represent intervaricose axon segments. Type II profiles, which were nominally larger than Type I structures, were characterized by a high density of round microvesicles, which were frequently concentrated along the neuronal membrane to form 'synaptoid' contacts with adjacent glia. Two additional and large GnRH profiles (>5 microm2) were observed. One (Type III) contained a high density of dcvs and mitochondria, and was considered analogous to an axonal swelling or Herring body in the magnocellular hypothalamo-neurohypophysial system. The Type IV structure, which was considered not to be a Herring body because of the relative low density of mitochondria was innervated by a classical symmetrical synapse. The functional significance of these observations is discussed.

Animals↗
PubMed2000

A technique for the evaluation of failed fallopian tube ligation with metal clips.

The evaluation of fallopian tubes after failed tubal ligation can be difficult because conventional histopathological techniques are unable to section the metal clips when in situ. Once the clips have been removed, any evidence of tube patency is lost. This report describes a technique of embedding and sectioning that enables sections to be made while the metal clips are still in situ. This is a modification of a method first described to embed mineralised bone and involves the use of plastic embedding and a diamond saw. Using this technique, a permanent record is made of the tube location and patency.

Adult↗
PubMed2000

Evaluation of LR white resin for histology of the undecalcified rat tibia.

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.

Acrylic Resins↗