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PubMed1989

Marked and variable inhibition by chemical fixation of cytochrome oxidase and succinate dehydrogenase in single motoneurons.

The effect of tissue fixation on succinate dehydrogenase and cytochrome oxidase activity in single motoneurons of the rat was demonstrated using a computer image processing system. Inhibition of enzyme activity by chemical fixation was variable, with some motoneurons being affected more than others. It was concluded that quantification of enzymatic activity in chemically fixed tissue provides an imprecise estimate of enzyme activities found in fresh-frozen tissues.

Animals↗
PubMed1992

Fixed-tissue micrographic surgery in the treatment of cutaneous melanoma. An overlooked cancer treatment strategy.

Many physicians have overlooked the value of adding zinc chloride tissue fixation prior to Mohs micrographic surgery, partly because of the absence of randomized clinical comparison trials. Nevertheless, historic control data indicate a survival benefit in the treatment of cutaneous melanoma. Three possible mechanisms are: 1) prevention of release of viable cells into circulation during surgery, 2) avoidance of disruption of clinically invisible satellite deposits, and 3) stimulation of a local inflammatory reaction and possible immune response.

Chlorides↗
PubMed1998

Ultrastructural in situ hybridization: a review of technical aspects.

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters; the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.

Animals↗
PubMed2001

A novel tracheal tissue connector for fixation of laryngeal prostheses.

A tissue connector (TC), basically consisting of a ring that will be integrated into the trachea, is under development to study the fixation of laryngeal prostheses. Two experiments have been performed to test the TC in goats. In experiment 1, a polypropylene mesh was implanted around the trachea. The meshes were explanted after 6 and 12 weeks. In experiment 2, the actual TC consisted of two titanium rings (inner ring and outer ring) executed as quarter rings, fixed on each other, and a polypropylene mesh like a sandwich in between. The titanium inner ring was implanted between two tracheal rings thus penetrating the trachea with the mesh around the trachea and the fixed titanium outer ring on the outside of the trachea. The TCs were removed after 12 weeks. Experiment 1 showed that the mesh was entirely infiltrated by host tissue. Inflammatory cells and high vascularisation were observed in 3 of 4 implants. However, in experiment 2, the mesh was completely incorporated by mature connective tissue without inflammation reaction. At some areas, deposition of cartilage tissue was observed. In conclusion, the TC was firmly embedded in the trachea thus being appropriate for its intended use.

Animals↗
PubMed1994

Microwave technology in diagnostic neuropathology.

The microwave oven has a large range of potential applications in diagnostic neuropathology, ranging from tissue fixation and processing for light and electron microscopy to immunocytochemistry and molecular neuropathology. This review highlights the major current applications for the microwave oven and emphasises areas of particular recent interest. Established microwave techniques are discussed and suggested modifications to published staining techniques are included. Future potential diagnostic applications of microwave technology are emphasised, particularly in terms of in situ hybridisation and immunocytochemistry. Although many microwave techniques for use in diagnostic neuropathology are clearly established, widespread use of the microwave oven has not yet occurred in most neuropathology laboratories. The reasons for this apparent delay and some occasional difficulties in the use of the microwave oven are discussed. Increasing clinical demand for rapid diagnosis (particularly with the use of stereotactic brain biopsy techniques in providing tissue specimens for surgical neuropathology) will enable neuropathologists and laboratory technical staff alike to reconsider the use of the microwave oven for many laboratory techniques and reassess the wide range of potential applications and benefits that this useful tool has to offer.

Animals↗
PubMedDate not supplied

Bioabsorbable implants in orthopaedics: new developments and clinical applications.

The use of bioabsorbable implants in orthopaedic surgical procedures is becoming more frequent. Advances in polymer science have allowed the production of implants with the mechanical strength necessary for such procedures. Bioabsorbable materials have been utilized for the fixation of fractures as well as for soft-tissue fixation. These implants offer the advantages of gradual load transfer to the healing tissue, reduced need for hardware removal, and radiolucency, which facilitates postoperative radiographic evaluation. Reported complications with the use of these materials include sterile sinus tract formation, osteolysis, synovitis, and hypertrophic fibrous encapsulation. Further study is required to determine the clinical situations in which these materials are of most benefit.

Absorbable Implants↗
PubMed1994

Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina.

The distribution of fibroblast growth factor (FGF) receptor in bovine retina was established using a polyclonal antibody against the extracellular domain of this receptor. Different conditions of tissue fixation and development of the secondary antibody were tested. The ability of the antiserum to map precisely the receptor was obtained on fresh-frozen sections which had been treated with paraformaldehyde prior to incubation with this antiserum. Positive staining was confined mainly to the synaptic and ganglion axon layers. These results suggest that the FGF receptor might act in the transmitter stability, and the plasticity of synapses and ganglion cell axons in adult retina.

Animals↗
PubMed2003

Toll-like receptor 2 is expressed by alveolar epithelial cells type II and macrophages in the human lung.

The ability of the host to recognize pulmonary invasion by pathogenic organisms and establish an appropriate host response to infection requires innate immune defense mechanisms. Early bacterial clearance in the lung is mediated by alveolar macrophages (AM) and polymorphonuclear neutrophils. Additionally alveolar epithelial cells type II (AEC-II) may act as immunoregulatory cells. The toll-like receptors (TLR) are part of this innate immune defense, recognizing conserved patterns on microorganisms. Toll-like receptor 2 (TLR2) is crucial in detecting components of gram-positive bacteria and mycobacteria. Signals initiated by the interaction of TLR2 with bacterial components direct the subsequent inflammatory response. The detection of TLR2 mRNA in human lung tissue prompted us to localize the expression of mRNA and protein at the cellular level using a novel method for tissue fixation. We utilized HOPE-fixed lung specimen sections for targeting mRNA by in situ hybridization and protein by immunohistochemistry using the monoclonal antibody TL2.1. In normal lung areas the expression of TLR2 mRNA and protein was found to be located in cells resembling AEC-II and AM. Expression of mRNA was verified by RT-PCR and DNA sequencing. These results indicate a potential mechanism of increased immunosurveillance at the alveolar level controlling the localized infection.

Epithelium↗
PubMed2003

Handling of clinical tissue specimens for molecular profiling studies.

The relationship between gene expression profiles and cellular phenotypes is an important aspect of functional genomics. Clinical tissue specimens will play a vital role in this effort. The usefulness of tissue for molecular profiling is significantly influenced by the manner of specimen handling. Crucial components of this process include the optimization of the methods of tissue fixation and embedding, not only to obtain excellent histological detail, but also to promote the elucidation of the gene and protein expression profiles. In this article, we describe handling of clinical specimens using whole-mount prostate as an example, the use of new high-throughput techniques that allow molecular profiling analysis and the use of a web-based 3-dimensional model to combine these data to make it available to clinicians and the research community. Complete protocols and additional discussion are available on the website, http://cgap-mf.nih.gov.

Cryopreservation↗
PubMed1998

Automated in situ hybridization: diagnostic and research applications.

Although in situ hybridization has been in use for almost 30 years, its technically demanding nature, the requirements for optimal tissue fixation and preservation, and the turnaround time for the experiments have prevented this technique from becoming widely used in the surgical pathology setting. The use of nonisotopic reporter molecules, the possibility of performing hybridization on archival material, and very recently, automation of the procedure have brought in situ hybridization to the forefront of diagnostic and experimental pathology. We describe our experience with nonradioactive, automated in situ hybridization, compare the technique with traditional manual procedures, and briefly outline its potential applications in diagnostic pathology and in the research setting.

Automation↗
PubMed1991

No evidence for effects of mild microwave irradiation on electrophysiological and morphological properties of cultured embryonic rat dorsal root ganglion cells.

Effects of mild microwave treatment (1 hr, 37 degrees C) on the in vitro development of rat mechanically dissociated dorsal root ganglion (DRG) neurons were investigated to establish whether microwave irradiation effects exist on nervous tissue other than heat induced tissue fixation. Phase contrast microscopy and immunocytochemical neurofilament stainings did not reveal significant differences between irradiated (2 hr after isolation) and control cultures, maintained up till 21 days. The electrophysiological properties of microwave exposed and non-exposed DRG neurons were compared using the whole-cell patch-clamp technique. Control neurons, in culture for 0-12 days, were excitable. In cultured cells (1-12 days), microwaved 2 hr after isolation, the action potentials were similar to or slightly different from those of the control cells. No acute microwave effects were found on neurons irradiated after 1 day of culture. These results suggest that mild microwave irradiation has neither significant acute nor strong long-term effects on DRG culture development and DRG neuron membrane properties, consistent with the notion that microwave effects essentially are temperature effects.

Action Potentials↗
PubMed1994

Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: relationship between the enzyme activity and immunointensity.

To understand changes associated with the menstrual cycle in the human ovary, it is very important to examine chronological changes in P450 aromatase (P450arom) enzymatic activity in the normal cycling ovary. Therefore, we initially examined the correlation between intensity of P450arom immunoreactivity and its biochemical enzymatic activity in five estrogen-producing human cancer cell lines (HHUA, Ishikawa, HEC-59, OMC-2, and MCF-7). P450arom immunointensity per cell was evaluated by the CAS 200 computed image analysis system, and its catalytic activity per 10(6) culture cells was analyzed by the tritiated water method. A significant correlation (r = 0.959) was demonstrated between P450arom immunoreactivity and enzymatic activity under optimal conditions of tissue fixation and immunohistochemical procedures. We then investigated P450arom immunointensity in 31 specimens of normal cycling human ovaries to examine chronological changes in P450arom activity per cell throughout the menstrual cycle. In the follicular phase, P450arom was observed in the granulosa cells of one selected antral follicle per case during the mid- to late proliferative period, and its immunointensity per granulosa cell in the follicle was not significantly different between mid- and late proliferative periods, although serum estradiol level was markedly elevated in the late proliferative period. In the luteal phase, both P450arom immunointensity per luteinized granulosa cell in a corpus luteum and serum estradiol level reached a peak in the mid-secretory period. These findings indicate that different factors may influence ovarian P450arom activity during the follicular and luteal phases, i.e., an increased number of granulosa cells in the selected follicle during the follicular phase but changes in P450arom activity per luteinized granulosa cell in the corpus luteum during the luteal phase.

3,3'-Diaminobenzidine↗
PubMed1994

[Antigen retrieval by microwave oven with buffer of citric acid].

Microwave oven (mwo) is used to stimulate tissue fixation and to retrieve antigens damaged by fixation. Heavy metal salt solutions, water, and citric acid buffer (cab) have been suggested for this purpose. A serie of tumors treated with cab and phosphate-buffered saline (pbs) with mwo were studied immunohistochemically with 24 antibodies. Controls were treated in the same way, except for microwaving. The antibodies were directed against antigens of the following tumors: breast and prostate carcinoma, carcinoid, lymphoma and melanoma. The results showed that cab enhanced the immunoreactivity of the following antigens: estrogen receptors (AMAC), progesterone receptors (Novocastra), HMB45, vimentin, leukocyte common antigen, PCNA, p53, MIB-1 (Ki-67) and prostatic specific antigen. The antigens that did not improve their immunoreactivity, when compared with the control series were: factor VIII, keratin, Leu 22, L26, neuron-specific enolase, CEA, chromogranin, HBME-1, smooth muscle actin and EMA. Microwaving equally improved protein S100 and desmin either with cab or pbs. The only antigen that improved with pbs was actin. The results with B72.3 and NKI/C3 were poor and not reliable. In conclusion microwaving with cab enhances the immunoreactivity of the antibodies mentioned above leading to an increase in sensibility without loosing specificity.

Antibodies, Monoclonal↗
PubMed1994

Establishment of the formalin-free surgical pathology laboratory. Utility of an alcohol-based fixative.

Concerns about the toxicity of formalin dictate the need for alternative methods of tissue fixation. Alcohol-based fixatives have been proposed as optimal for immunohistochemical and nucleic acid methods and may be useful for diagnostic light microscopy. We recently converted our surgical pathology laboratory from 10% neutral buffered formalin to an alcoholic fixative (56% ethanol and 20% polyethylene glycol) and compared 100 consecutive cases after conversion with 100 formalin-fixed cases prior to conversion. One representative slide from each case was reviewed independently by four pathologists without knowledge of the fixative employed. The quality of fixation of each case was graded (0, very poor; 1, below average; 2, average; and 3, above average) based on the subjective composite evaluation of seven morphological features: tissue architecture, cell borders, cytoplasm, nuclear contours, chromatin texture, red blood cell membranes, and uniformity of staining. No significant difference in the quality of fixation was observed, although the average score of the alcoholic fixative was slightly lower than that of the formalin (2.59 vs 2.74). Interobserver concordance was 74%. Advantages of the alcoholic fixative included elimination of carcinogenic vapors, more rapid fixation, greater stain avidity, elimination of enzyme predigestion in immunohistochemical studies, and simple and rapid disposal. Disadvantages included slightly increased viscosity, increased variability of tissue staining and nuclear shrinkage, artifactual pigment deposition in bloody specimens, and increased flammability risk. Alcohol-polyethylene glycol appears to be a satisfactory alternative to formalin in routine diagnostic surgical pathology.

Ethanol↗
PubMed1984

Distribution of exudated FITC-dextrans in experimental vasogenic brain edema produced by a focal cryogenic injury.

Mice were subjected to cortical cryogenic brain injury, and FITC-dextrans (mol. wt. 20,000 or 150,000) were injected intravenously (i.v.). After a survival period of 4 h the distribution of the FITC-dextrans was determined by a histotechnical procedure described recently ( Hultstr öm et al. 1982a ). This technique is based on freeze-drying and vapor fixation to immobilize the tracer and to provide tissue fixation. In and around the cryogenic injury both tracers leaked out of the cortical and the leptomeningeal vessels and spread into the brain parenchyma. They were seen as multiple, closely apposed droplets of fluorescent material best recognized by fluorescence microscopy under high magnification. The tracers were also taken up by neuronal perikarya and in glial cell nuclei of, presumably, astrocytic origin. Our study shows that the FITC-dextran technique can be used for experimental studies on the vasogenic form of brain edema. The patterns formed by the extravasated tracers have qualitative similarities to those produced by other more commonly used tracers, such as fluorochrome-labeled serum proteins and peroxidase.

Animals↗
PubMed2004

Three-dimensional visualization analysis of in vitro cultured bone fabricated by rat marrow mesenchymal stem cells.

Marrow mesenchymal stem cells are well known for their differentiation into bone-forming osteoblasts and in vitro mineralized tissue formation. However, process details, including tissue structure and cellular environments, remain unclear. The present study demonstrates three-dimensional visualization of tissue fabricated by culturing MSCs in the presence of calcein, a fluorescent marker for bone mineralization. The 3D visualization was performed by computer-assisted confocal laser scanning microscopy and revealed that the in vitro tissue consisted of layers of a mineralized matrix with round cells in the matrix lacunae, an unmineralized matrix (osteoid), and osteoblastic cells on the osteoid surface. The findings show that the mineralization by cultured MSCs is an in vitro counterpart of in vivo bone formation and indicate that the novel technique of visualization without tissue fixation could be useful for continuous monitoring of tissue organization in an ongoing culture.

Alkaline Phosphatase↗