[THE EMBEDDING OF ORGANS OR ANATOMICAL SPECIMENS IN TRANSLUCENT PLASTICS].
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Anatomy↗
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Kinetics of bromodeoxyuridine uptake by smooth muscle cells after arterial injury.
The time course of 5-bromo-2'-deoxyuridine (BrdU) uptake by medial and neointimal smooth muscle cells was examined in rat carotid arteries at periods of 1-20 days after balloon catheter injury. DNA-incorporated BrdU was determined by the indirect immunoperoxidase technique in both plastic- and paraffin-embedded specimens. The BrdU labeling index (LI) peaked at 48 h in the media (19.9% paraffin, 9.4% plastic) and at 5 days in the neointima (55.4% paraffin, 60.2% plastic). Immunohistochemistry for the endogenous marker proliferating cell nuclear antigen was variable and generally less reliable than BrdU. Using total viable medial smooth muscle cell number as an index of medial injury, the LI for BrdU was directly proportional to the extent of injury at 48 h, and correlated with loss of actin immunoreactivity. BrdU immunohistochemistry is a reliable alternative to [3H]-thymidine autoradiography for short-term labeling in this model. Proliferation of smooth muscle cells in this model is likely related to the degree of injury to the media.
Actins↗
Peg fracture in a Tricon P tibial component.
Revision of a total knee replacement because of medial tibial plateau insufficiency revealed the base plate of a noncemented plastic-peg tibial component to have sheared off the plastic pegs which were still tightly embedded in bone. The fact that these plastic pegs remained tight indicates that even in extremely adverse situations the pegs are capable of providing fixation.
Aged↗
Combined lectin binding and PAS/alcian blue staining in glycol methacrylate sections.
Evaluation of cryofixation and paraffin and glycol methacrylate embedding showed that lectin binding was essentially independent of the embedding medium. Fluorescence intensity increased in the following order: glycol methacrylate, paraffin and cryostat sections. The optical resolution increased in the reverse order. Semi-thin glycol methacrylate sections provided satisfactory fluorescence intensities and the best resolution of all embedding techniques applied. Furthermore the lectin treated sections can be stained further using routine histological or specific histochemical methods. The potassium hydroxide/alcian blue/periodic acid-phenylhydrazine-Schiff method was used successfully to demonstrate sulfated and nonsulfated sialomucins. Lectins combined with mucin histochemistry allowed visualization of specific sugar residues in the same glycol methacrylate plastic section.
Alcian Blue↗
Demonstration of platinum microcoils in embolized blood vessels in-situ using a modified methyl methacrylate embedding method, and a special cutting and grinding technique.
With current paraffin embedding techniques it is not possible to demonstrate metal particles in vascular lumens. We used a modified methyl methacrylate embedding method for obtaining plastic blocks which were cut with a precision diamond saw, and subsequently ground and polished to preserve the interface of reactive tissue surrounding platinum microcoils in arteriovenous malformations. Artifact-free interfaces could be achieved by slow embedding under vacuum conditions, slow polymerization in a water bath, and very gentle cutting, grinding and polishing techniques. The method produces good preservation of tissue and cells directly adjacent to metal coils. Our method is useful for the histomorphological interpretation of vascular pathologies in which metal particles have been introduced.
Adult↗
Reversible embedment cytochemistry (REC): a versatile method for the ultrastructural analysis and affinity labeling of tissue sections.
Reversible embedment cytochemistry (REC) is a new method for revealing cellular ultrastructure and for improving access of intracellular targets to macromolecular affinity labels. Fully polymerized polymethylmethacrylate was dissolved in dichloromethane and infiltrated into fixed tissue-culture cells and tissues. After evaporation of the solvent, samples were left in hard plastic. Samples were thus embedded without exposure to chemical polymerization reactions that might damage tissue ultrastructure or antigenicity. Glass or diamond knives fitted with water troughs were used to cut sections 30-1000 nm thick. Since polymethylmethacrylate is composed of linear polymers that are not covalently crosslinked, the plastic was easily extracted from the sections by immersion in solvent. Subsequently, various preparative methods, including negative staining, critical point-drying, and platinum-carbon rotary shadowing, were used to provide detailed images of well-preserved cell structure for conventional and high-voltage transmission electron microscopy. Fluorescein-conjugated affinity labels were used to obtain subcellular distributions of target molecules in semi-thick sections of cultured cells and tissues for light microscopy. Colloidal gold-labeled antibodies were used to localize microtubules in sections of cultured cells by electron microscopy. REC is a versatile method that should find wide application in many studies of cellular function.
Affinity Labels↗
Estimating the effect of etching agents on plastic sections.
The effects on epoxy embedding media of certain etching procedures have been studied in two ways. Firstly the resin present in thick sections, before and after etching, was demonstrated by staining the plastic with Sudan Black B. Secondly the change in weight of plastic blocks after their exposure to etching procedures was measured. Methods involving halogens in acetone, alkoxides in ethanol or in methanol-benzene, and performic acid resulted in gross removal of plastic. Additionally, in keeping with the widespread belief that etching may increase permeability of plastic sections to stains, most of the etch methods studied, including organic solvents alone, did remove some plastic from the blocks.
Epoxy Resins↗
[Embedding of undecalcified bone tissue for electron microscopic investigation (author's transl)].
UNLABELLED: The preparation of undecalcified bone tissue for electron microscopic investigation depends on the quality of the embedding medium. Penetration of bone tissue is improved by a low viscosity of the monomer plastic material. Hardness of polymerized embedding medium should be similar to bone tissue, but a good cutting quality must be maintained. A low viscosity resin proposed by Spurr (1969) proved to be in compliance with these conditions. MATERIAL AND METHODS: The embedding medium is composed of four components: vinylcyclohexenedioxide (ERL, 4206), polyglycolepoxide (DER 736), nonenyl succinic anhydride (NAS) and dimethylaminoethanol (DMAE). The most favourable molar ratio between anhydride (NSA) and epoxide (ERL 4206 and DER 736) was A : E = 0.81 : I. Hardness and brittleness of polymerized epoxy resin can additionally be influenced by varying the amounts of both epoxy components in the final mixture. Human iliac crest trabecular bone as well as rat bone tissue were embedded to test the quality of the medium. Resutls: Ultra-thin sections were performed at low cutting speed (0.5 mm/sec) using diamond knives. Artificial alterations of bone surfaces did hardly occur due to a good penetration of the bone tissue. The special hardness of the embedding medium stabilized the tissue, thus chattering of the transition from osteoid to mineralized bone could be markedly reduced.
Anhydrides↗
Sterile shunt malfunction. A scanning electron microscopic perspective.
Recently, a delayed hypersensitivity reaction to silicone plastics has been demonstrated in some patients with breast and joint implants and subcutaneously injected silicone. In this study, the authors examined the internal surface of shunting systems to evaluate the cellular response to implanted silicone plastic. The distal peritoneal tubing from 20 patients with ventriculoperitoneal shunts was examined with a scanning electron microscope. Twelve tubes were removed because of documented distal shunt malfunction and eight for an elective lengthening procedure. Cultures of all tubing were negative. The catheters removed for malfunction contained a variety of cells: sometimes in clumps, sometimes associated with platelets and densely adherent to the silicone tubing wall. In several shunts, giant cells were seen with multiple foot processes adherent to the internal silicone surface. The internal surface of the tubing of two malfunctioning shunts was embedded in electron microscopy plastic and studied with transmission electron microscopy. The cells proved to be neutrophils with no evidence of silicone granules inside the cell bodies. The shunts removed for elective revision showed only a few cells, and those were never associated with platelets. There was proteinaceous material scattered on the internal surface of the tubing, but the cellular response was markedly different from that in malfunctioning shunts. The authors postulate that the findings in malfunctioning shunt tubing represent a delayed hypersensitivity to silicone similar to that seen with other types of silicone implants.
Adolescent↗
Microwaves for immunohistochemistry.
Microwaves are now widely used in immunohistochemistry for fixing and stabilizing tissue prior to embedding and cutting, for antigen retrieval and for immunoincubations. These techniques can be used for frozen sections and for material embedded in paraffin and plastic. Material prepared in this way shows high contrast in light microscopy. In principle, these microwave methods can also be used for electron microscopy. To be successful in the application of these techniques, insight into the physics of exposure to microwaves and the effects of microwaves on the material is a must. Microwave immunohistochemistry depends on optimal temperature control. To guarantee this, special measures should be taken and dedicated laboratory ovens should be used. The recently developed Coverplate units facilitate immunoincubations in the microwave oven. We show that the total microwave approach, combining microwave fixation, embedding and immunoincubations, is very useful for confocal microscopy.
Antigens↗
Wild carnivore acceptance of baits for delivery of liquid rabies vaccine.
A series of experiments are described on the acceptance, by red foxes (Vulpes vulpes) and other species, of two types of vaccine-baits intended to deliver liquid rabies vaccine. The baits consisted of a cube of sponge coated in a mixture of tallow and wax, or a plastic blister-pack embedded in tallow. All baits contained tetracycline as a biological marking agent: examination of thin sections of carnivore canines under an ultraviolet microscope revealed a fluorescent line of tetracycline if an individual had eaten baits. Baits were dropped from fixed-wing aircraft flying about 100 m above ground at approximately 130 km/h. Flight lines followed the edges of woodlots midway between parallel roads. Baits were dropped at one/sec, resulting in one bait/36 m on the ground, or 17 to 25 baits per km2. Crows (Corvus brachyrhynchos) removed many baits, but did not appear to lower the percent of the fox population which took bait. Dropping baits only into corn and woodland to conceal baits, to reduce depredation by crows, reduced acceptance by foxes. Acceptance by foxes ranged between 37 and 68%. Meat added as an attractant did not raise acceptance. Presence, absence, color and perforations of plastic bags did not alter bait acceptance. Dispersal by juvenile foxes probably lowered the estimates of bait acceptance. It took 7 to 17 days for 80% (n = 330) of foxes to eat their first bait. The rapidity with which foxes picked up their first bait appeared more affected by unknown characteristics of years or study areas than by experimental variables. Skunks (Mephitis mephitis) and raccoons (Procyon lotor) also ate these baits, but acceptance was lower. Small mammals contacted baits, but rarely contacted the vaccine, which had the potential for vaccine-induced rabies in some species. Aerial distribution of baits was more cost-effective than ground distribution as practiced in Europe. This system has potential for field control of rabies, although higher acceptance will be desirable.
Animals↗
Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold.
Ultrastructural localization of growth hormone in rat anterior pituitary and of muscle-specific actin in rabbit arterial smooth muscle cells was accomplished with a post-embedment procedure using colloidal gold. Plastic sections (2 microns) were mounted on slides, deplasticized, immunostained with immunoglobulin-colloidal gold particles, re-embedded in Epon, and sectioned for electron microscopy. This procedure enabled light and electron microscopic localization of these intracellular antigens on the same section. Positive immunostaining was demonstrated with this procedure with a muscle-specific actin antibody which previously failed to localize antigenic sites by EM. The procedure described yielded staining of high specificity, with minimal background and well-preserved ultrastructure. This re-embedding technique is useful in situations where problems with post-embedding EM immunostaining exist and where correlative LM and EM immunostaining is essential.
Actins↗
Protein synthesis by rat hippocampal slices maintained in vitro.
The present study evaluates protein synthesis in rat hippocampal slices maintained in vitro. Transverse slices of hippocampus were prepared from both adult rats and rat pups during postnatal development and incubated in a gassed (95% O2/5% CO2) balanced salt medium containing 5 nM 3H-leucine. The time course of 3H-leucine incorporation into TCA-precipitable protein was determined using slices removed from the media after 5, 10, 20, 30, 40, 60, and 120 min of incubation. The pattern of 3H-amino acid incorporation was evaluated by fixing slices with paraformaldehyde, embedding the slices in plastic, and sectioning the slices end on and en face for autoradiographic analysis. Biochemical analysis of 300 and 400 micron slices revealed that incorporation of leucine into protein proceeds at a constant rate. The autoradiographic analysis revealed that in adult hippocampal slices of 300-600 micron thickness there was complete penetration of 3H-leucine with no indication of a gradient in the extent of incorporation throughout the slice. The pattern of grain density within 300-600 micron slices matches that previously reported after in vivo injections of radiolabeled amino acid, where grain density is highest over neuronal cell bodies and lower over the laminae that contain dendritic processes and axons (Phillips et al: Mol Brain Res 2:251-261, 1987). Hippocampal slices of 200, 800, and 1,000 micron thickness showed irregular labeling. Slices of 200 micron were filled with pyknotic nuclei and vacuoles and exhibited patchy labeling. In 800 micron slices there were isolated areas of good preservation within the slice core, but these areas exhibited little incorporation. Relative to the 300-600 micron slices, there was a higher number of pyknotic nuclei and a much deeper layer of necrosis along the cut edges. Slices of 1,000 micron thickness showed poor preservation throughout and low levels of incorporation. Biochemical studies revealed a much higher rate of incorporation in the slices prepared from postnatal animals. Autoradiography of the slices from developing rats revealed that penetration was excellent and incorporation appeared to be greater as judged by an overall higher grain density. We believe that rat hippocampal slices provide a good in vitro model of protein metabolism that will be useful for studies of protein synthesis in isolated cell body and dendritic laminae and for the evaluation of whether protein synthesis in particular laminae is regulated by synaptic activity.
Amino Acids↗
The "practical mathematics" of recording three-dimensional eye position using scleral coils.
The "gold standard" for recording the three-dimensional rotation of the eye involves placing two coils of wire, embedded in a soft plastic ring, on the sclera of the eye, then placing the subject inside a set of orthogonal oscillating magnetic fields, and using the currents induced in the eye coils to deduce the position of the coil, and hence of the eye, in space. Eye movements are actually eye rotations, which can be described mathematically by a special class of matrices, rotation matrices, or, alternatively, by a rotation vector related to the axis of the rotation. This article deals with the mathematical tools needed to implement the signal processing from such a multifield, dual-coil system and compute the precise rotational movement of the eye. One reason for making such careful measurements is to study an interesting constraint on eye movements, called Listing's law, which expresses ocular torsion, or rotation of the eye about its line of sight, in terms of the direction of gaze. Techniques for experimentally quantitating these constraints are also presented. Following a treatment of the "ideal" case, with coils and eye in perfect alignment, the additional techniques for dealing with various departures from ideality that are almost always encountered experimentally are examined. A final section deals with developing a validation protocol for eye movement analysis techniques using mechanical and computer simulations of eye movements.
Algorithms↗
The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background.
The classical von Kossa method has been modified: the high silver nitrate concentration in the original was replaced by 0.05% silver lactate with hydroquinone remaining the reducing agent of choice. The present modification stained calcification nodules with a sensitivity comparable to the original von Kossa reaction, but resulted in a reduced background staining in cultured osteoblasts. The method works well also with plastic- or paraffin-embedded tissue sections.
Calcification, Physiologic↗
The anabolic effect of estrogen on endosteal bone formation in the mouse is attenuated by ovariohysterectomy: a role for the uterus in the skeletal response to estrogen?
In the mouse, the anabolic effect of estrogen on the uterus and its stimulatory effect on endosteal bone formation are well documented. When these observations are coupled with the recent description of uterine-derived bone cell mitogens, it raises the possibility that uterine hypertrophy in response to estrogen might lead to the production and release of factors that participate in the skeleton's anabolic response to estrogen. To determine if the stimulatory effects of estrogen on endosteal bone formation and uterine tissue in the mouse are related, we have studied this specific skeletal response to ovariectomy (OVX) and ovariohysterectomy (OHTX), and to two levels of 17 beta-estradiol (17 beta-E2). To assess treatment effects, 48 Swiss-webster mice were assigned to six groups: OHTX/oil vehicle, OVX/oil vehicle, OHTX/150 micrograms 17 beta-E2, OHTX/300 micrograms 17 beta-E2, OVX/150 micrograms 17 beta-E2, and OVX/300 micrograms 17 beta-E2. Animals were treated once per week with vehicle or the respective 17 beta-E2 dose. To quantitate bone formation, fluorochrome labels were administered at the beginning and end of the experimental period. At the conclusion of the 5-week study, tibiae were processed undecalcified for embedding in methyl methacrylate plastic. Cross-sectional areal properties and bone formation rates were quantitated from 30 microns mid-diaphyseal sections using a Bioquant Bone Morphometry system. Compared with the vehicle-treated OVX and OHTX mice, 150 micrograms of 17 beta-E2 administered once per week significantly increased cortical bone areas (P less than 0.05) but cortical bone widths and the ratio of cortical bone area to total bone area was increased only in estrogen-treated OVX mice (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Monoclonal antibodies against calcitonin. Characterization and application in light and electron microscopy immunocytochemistry.
Twenty-seven monoclonal antibodies (MAbs) to synthetic human calcitonin (CT) were characterized for their reactivities with human CT peptide fragments by dot-blot analysis on nitrocellulose paper. Most of the antibodies bound to the C-terminus and fewer to the mid-region of CT. We have studied thyroid tissue specimens from several animal species after fixation in paraformaldehyde-, glutaraldehyde- or picric acid-containing mixtures and cryostat sectioning or embedment in paraffin or plastic (Epon 812 or Lowicryl 4KM) using this panel of MAbs. The site of antigen-antibody reaction was revealed either by immunoperoxidase, immunoalkaline phosphatase or by silver-enhanced immunogold staining methods. All MAbs were able to localize CT in human, rat and mouse thyroid C cells. Nineteen MAbs recognizing synthetic salmon CT and synthetic [Asu1,7]-eel CT by dot-blot, reacted with chicken ultimobranchial body C cells. One MAb recognizing native porcine CT by dot-blot, stained C cells in hog thyroid. Immunopositivity was confined to the cytoplasm and ultrastructural immunogold labelling demonstrated that cytoplasmic secretory granules were stained. Surgical specimens from human medullary thyroid carcinoma were also analysed for the presence of CT and a variable number of positive cells was found. Furthermore, Congo red-positive areas were shown to react with the MAbs. All conventional staining and immunoabsorption controls were negative. Hence, these MAbs may be suitable for use in routine immunopathological diagnosis of CT-producing tumors and for immunocytochemical localization of the three major CT variants in different animal species.
Animals↗
Cytochemical analysis of oligosaccharide processing in frog photoreceptors.
Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections of Xenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with the N-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.
Animals↗