PLASTIC EMBEDDING MIXTURES FOR USE IN ELECTRON MICROSCOPY.
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Electrons↗
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A new method for identification of cement lines in undecalcified, plastic embedded sections of bone.
A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with osteochrome before plastic embedding. After sectioning at 5 microns on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-24 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 micron wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 microns wide. The method also demonstrates abnormal halo volumes around osteocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease.
Bone Diseases↗
Cerium-based demonstration of phosphatase activity in plastic-embedded sections: a comparison with conventional methods.
Cerium-based methods have been used for the demonstration of several phosphatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-embedded sections. At alkaline pH calcium is used as capturing agent and the precipitated calcium phosphate converted to cerium phosphate. At neutral and acid pH cerium is used directly as capturing agent. Cerium phosphate is subsequently visualized using the H2O2-DAB method. A comparison has been made with conventional calcium-cobalt and lead methods. It appeared that calcium-cobalt methods are more susceptible to improvement than lead methods.
5'-Nucleotidase↗
Induction of a heat shock gene (hsp70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue.
Elevation of body temperature by 2-3 degrees C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.
Animals↗
Cellular localization of heat shock gene expression in rabbit cerebellum by in situ hybridization with plastic-embedded tissue.
The major cell types in rabbit cerebellum which engage in the expression of a heat shock gene (hsp70) after hyperthermia were identified. This required in situ hybridization on thin sections derived from plastic-embedded tissue. All classes of cerebellar neurons which were examined (Purkinje, granule, and stellate cells) responded by induction of hsp70 mRNA within 1 hr after hyperthermia. Prominent induction of hsp70 mRNA was also observed in oligodendroglia in the deep white matter.
Animals↗
Progress in temporal bone histopathology. I. Semithin 3-5 micron sectioning of undecalcified human temporal bone after plastic embedding.
Prior to embedding a human temporal bone in methylmethacrylate the plane of sectioning is determined through CT-scanning. A new technique of micro-slicing, plastic embedding and semithin sectioning of undecalcified human temporal bone is described.
Humans↗
Thionin staining of paraffin and plastic embedded sections of cartilage.
The usefulness of thionin for staining cartilage sections embedded in glycol methacrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin O stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin O, thionin did not show metachromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.
Aged↗
Mycosis fungoides: initial diagnosis via palatal biopsy with discussion of diagnostic advantages of plastic embedding.
The authors present an example of mycosis fungoides which was initially diagnosed from a palatal biopsy. The distinctive nuclear morphology of the tumor cells, with a discussion of their diagnostic importance, is presented. The advantages of plastic-embedded formalin-fixed tissue are delineated.
Aged↗
Simultaneous demonstration of alkaline and acid phosphatase activity in bone, at bone-implant interfaces and at the epiphyseal growth plate in plastic-embedded undemineralized tissues.
The aim of the present study was to determine if it was possible to detect in bone, at the epiphyseal growth plate and at the bone-implant interface, the presence of alkaline and acid phosphatases using Technovit 7200 VLC resin. In the plastic-embedded specimens it was possible to observe the simultaneous presence of acid and alkaline phosphatases at the epiphyseal growth plate in the presence of intra-articular implants. The morphology of the cells positive for the phosphatases was very clear, with no apparent diffusion of the reaction product and no sputter ground staining.
Acid Phosphatase↗
Morphometry, densitometry and pattern analysis of plastic-embedded histologic material from urothelial cell carcinoma of the bladder.
An image analysis method of grading histologic sections of bladder carcinoma was tested. The method was new in four respects. First, for fixation of the biopsies a coagulant fixative was used. Second, 2-microns plastic sections were used to ensure the reproducibility of nuclear imaging. Third, a new stereologic approach was used for calculation of the nuclear volume and DNA content. Fourth, for the classification rule the morphometric, densitometric and texture features were used in concert. The IBAS 2000 instrument was used for the measurements. Texture analysis of the chromatin patterns was performed using Markovian texture features. Using discriminant analysis, of 22 parameters, 2 morphometric, 2 densitometric and 3 texture features were selected for the classification rule. With them, 89% of the bladder carcinomas were correctly classified into the three grades. All grade III tumors were classified correctly. Among the features tested, the densitometry of the DNA had the highest F values. All of the grade III tumors and 45% of the grade II tumor group had DNA histograms indicating aneuploidy. This study showed that plastic-embedded material is well suited to morphometry and densitometry and can be used for quantitative grading of bladder carcinoma.
Carcinoma, Transitional Cell↗
Stainable bone iron in undecalcified, plastic-embedded sections. Occurrence in man related to the presence of "free" iron?
Iron demonstrable with the Prussian blue reaction at the osteoid/mineralized tissue interphase (osteoid seam) of trabecular bone was observed in only 2.3% of a total of 1536 conventionally fixed and processed, undecalcified, plastic-embedded biopsy specimens taken from the iliac crest of patients for various diagnostic purposes. In marked contrast, clearly stainable bone iron was noticed in all 4 biopsy specimens from the iliac crest and in 11 of 15 vertebral bone fragments obtained at autopsy from individuals with verified primary or secondary hemochromatosis. Findings, including results obtained in vitro, suggest that a positive Prussian blue reaction at the surface of trabecular bone signals the presence of low-molecular-weight ("free") iron, which can bind to the osteoid matrix directly, ie, without the help of osteoblasts. Stainable bone iron may thus be a useful criterion for early detection of hemochromatosis and other types of potentially toxic iron overload.
Adolescent↗
Bone metastases of differentiated and medullary thyroid gland carcinomas. Usefulness and limitations of immunohistology performed on undecalcified plastic-embedded tissue specimens.
Undecalcified methylmethacrylate(MMA)-embedded biopsies and surgical specimens from 20 bone metastases of differentiated or medullary thyroid carcinomas or prostate carcinomas were investigated immunohistologically for the presence of thyroglobulin, cytokeratin, vimentin, and CEA. The immunoreactions on MMA-sections revealed the same staining patterns as those demonstrated using paraffin sections of the primary lesions. Conversely, immunohistological examination of decalcified paraffin-embedded specimens of the same metastases yielded either false-negative results or results that did not allow an exact evaluation. The findings demonstrate the usefulness and limitations of immunohistology when performed on undecalcified plastic-embedded material.
Adult↗
Neuronal ceroid lipofuscinosis. Diagnosis by semithin plastic-embedded sections of peripheral blood lymphocytes from a patient with a normal blood smear.
Neuronal ceroid lipofuscinosis is an important diagnostic consideration in a school-aged child with progressive blindness. Neurologic symptoms and signs may appear late, and the retinopathy is not specific. Ultrastructural abnormalities have been demonstrated in many body tissues, but skin has been claimed to be the most reliable. Light and electron microscopic examinations of WBCs have also been helpful in several instances. In the case reported, the skin biopsy specimen and light microscopy of a peripheral blood buffy coat failed to disclose diagnostic changes. Examination of semithin sections of plastic-embedded peripheral blood provided the answer.
Capillaries↗
Propionibacterium acnes cerebral botryomycosis. The role of plastic embedding in the diagnosis of grain-producing infections.
Botryomycosis is a chronic infection in which bacterial colonies, or "grains," occur in purulent exudate associated with draining sinus tracts or abscess formation. This condition usually is caused by Staphylococcus and Streptococcus, but other organisms occasionally have been implicated. This report describes the first case of botryomycosis caused by Propionibacterium acnes, a diphtheroid that has been rarely associated with opportunistic disease. In this case, plastic embedding allowed the observation of the structural details of the organisms composing the grains and permitted their presumptive identification as diphtheroids. The differential diagnosis of infectious agents that produce grains in tissue sections also will be discussed.
Adult↗
Double immunohistochemical demonstration of antigen expression and DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections.
In this study a double immunohistochemical staining procedure is described for the simultaneous visualization of antigen expressing cells and replicating cells. Cell surface antigen expression was marked with a monoclonal antibody against I a (His 19) or a monoclonal antibody against a membrane component of the cells of the monocyte-macrophage lineage (ED2). Replicating cells were detected by the incorporation of 5-bromodeoxyuridine. The method was applied sequentially. On frozen sections two peroxidase labeled reagents were used with two different substrates yielding a red and a dark-blue black reaction product. On plastic-embedded sections a peroxidase and an alkaline phosphatase labeled reagent were applied resulting in a brown and a blue reaction product.
Animals↗
Understanding microwave-stimulated Romanowsky--Giemsa staining of plastic embedded bone marrow.
Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky--Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky--Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.
Animals↗
Plastic embedding of undecalcified bone.
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Bone and Bones↗
Plastic embedding of cleared human fetuses.
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Fetus↗