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PubMed1986

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives.

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.

Antibodies, Monoclonal↗
PubMed1983

Endoscopic control of gastrointestinal hemorrhage by local injection of absolute ethanol: a basic assessment of the procedure.

To investigate the hemostatic mechanism of local ethanol injection, ethanol was injected into the gastric mucosa of five adult mongrel dogs and one guinea pig and histologic changes at acute and healing stages were followed. Following a local injection of absolute ethanol, the blood flow in small vessels at the site of injection became instantaneously arrested. Histopathologically, thrombosis of blood vessels in the mucosa and submucosa with edema, predominantly submucosal, was found 10 min after the ethanol injection. There was little or no inflammatory cell infiltration in the injected region. By 4 days after the injection, the base of the formed ulcer became stabilized with a uniform white coating, which, microscopically, was mainly composed of a thick layer of necrotized mucosal tissue. Ethanol showed fixative activity when applied to tissues at concentrations of greater than or equal to 20% although an ethanol concentration of at least 70% was required to accomplish adequate tissue fixation. With 95% or lower concentrations of ethanol, dehydration of tissue was insufficient, and hemostasis due to local vasoconstriction was less conspicuous than with absolute alcohol. Nevertheless, a fixative effect with consequent degeneration and necrosis of cells and secondary thrombosis was evident even at these ethanol concentrations. In patients treated with this hemostatic procedure, exposed blood vessels in the gastric ulcer became necrotized and corroded by fixation with injected absolute ethanol and disappeared from the base of the ulcer within 24 hr as seen in the animals.

Adult↗
PubMed1982

hard tissue biochemistry: a comparison of fresh-frozen and formalin-fixed tissue samples.

Formalin fixation of hard tissues alters the levels of some, but not all, of the parameters routinely measured by bone researchers. Although mineral parameters (ash weight, particle size (beta 1/2 002) and Ca/PO4 ratio) and lipid parameters (total lipid content and composition, extractability, and Ca-acidic phospholipid phosphate content) are not affected by formalin fixation, the uronate and DNA contents are reduced in formalin-fixed bone while hydroxyproline content is elevated.

Bone and Bones↗
PubMed2006

Bioabsorbable screw divergence angle, not tunnel preparation method influences soft tissue tendon graft-bone tunnel fixation in healthy bone.

Bone tunnel fixation of a soft tissue tendon graft is the weak link immediately following ACL reconstruction. This biomechanical study evaluated the influence of extraction drilled or step dilated bone tunnels and bioabsorbable screw divergence on soft tissue tendon graft fixation. From an initial group of 50 available specimens, similar apparent bone mineral density porcine tibiae (1.2 +/- 0.24 g/cm2) were divided into two groups of ten specimens each. Group 1 (extraction drilled) received 9 mm diameter tunnels. Group 2 (step dilated) received 7 mm diameter tunnels that were dilated to 9 mm. Grafts were secured in tunnels using 10 mm diameter, 35 mm long tapered screws. After high resolution CT scanning to evaluate screw divergence, constructs were pretensioned on a servo hydraulic device between 10 and 50 N for 10 cycles, and isometric pretensioned at 50 N for 1 min, prior to 500 sub-maximal loading cycles (50-200 N) and load to failure testing at 20 mm/min. Wilcoxon Signed Rank tests and Mann-Whitney U-tests were used to evaluate group differences. Coefficient of determination values (r2) were calculated to further delineate statistically significant relationships. Tunnel preparation method did not display statistically significant effects on insertion torque, displacement during cyclic testing, relative stiffness during cyclic testing, load at failure, stiffness during load to failure testing or displacement during load to failure testing. Screw divergence < 15 degrees produced lower displacement and greater relative stiffness during cyclic testing and greater load at failure and stiffness during load to failure testing. Screw divergence angle displayed moderate relationships with construct displacement during cyclic testing (r2 = 0.54), stiffness during load to failure testing (r2 = 0.60), and load at failure (r2 = 0.41). Tunnel dilation does not enhance soft tissue tendon graft fixation strength in healthy bone. Bioabsorbable screw divergence of > or = 15 degrees significantly reduces soft tissue tendon graft-bone tunnel fixation.

Absorbable Implants↗
PubMed1995

[External fixation for support in soft tissue reconstruction in hand surgery].

A mini fixator system was used to assist the operative correction of soft-tissue flexion contractures. The external fixator served as a tool for gradual correction and to secure joint position after soft-tissue release. Following wound healing, certain fixator components were unlocked and joint mobilisation initiated. Correction involved a contraction of the first web-space as well as flexion contractures of the index, middle, ring, and small fingers. The fixators were removed after four to six weeks. Complications related to the fixator equipment were not observed. The spread from thumb to index finger tip was increased from 3.5 to 16.5 cm. The functional result in sustained grip strength reached a value of 82% compared to the uninjured opposite side.

Adult↗
PubMed1980

Microwave fixation of cells in tissue culture.

Use of microwave irradiation has been evaluated for the in situ fixation of cells grown in tissue culture prior to fluorescent antibody staining. The results show total retention of cell protein in the matrix with microwave fixation, whereas 40-50% of the protein is lost during conventional formaldehyde fixation. Fluorescent antibody staining of cells shows that this protein loss reflects cell loss. A further advantage of microwave fixation is that photometric quantitation of fluorescence appears possible by random scanning of fluorescent antibody stained populations.

Cell Adhesion↗
PubMed2001

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

Ammonia↗
PubMed1996

Effect of fixation pressure on juxtacanalicular tissue and Schlemm's canal.

PURPOSE: A quantitative study was performed to compare the effect of two commonly used fixation pressures, 0 mm Hg (immersion) and 10 mm Hg (perfusion), on the porosity of the juxtacanalicular tissue and the size of Schlemm's canal. METHODS: Twelve pairs of human eyes were studied by fixing one eye with perfusion fixation and the fellow eye with immersion fixation. Morphometric analysis of the juxtacanalicular tissue and Schlemm's canal was performed. Outflow resistance was calculated from these measurements and compared with the measured outflow resistance obtained in six eyes. RESULTS: Schlemm's canal was narrowed in perfusion-fixed eyes, with a 47% smaller cross-sectional area than in immersion-fixed eyes (P = 0.04). Juxtacanalicular tissue of perfusion-fixed eyes had a 13.4% increase in the relative amount of empty space when compared with immersion-fixed fellow eyes (P = 0.04). Solid tissue components were almost equally divided among amorphous basement membrane, tendon and sheath material, and cytoplasm. No obvious washout of extracellular material was noted in perfused tissue. Measured outflow resistance was 100 times larger than outflow resistance of the juxtacanalicular tissue calculated from histologic measurements. CONCLUSIONS: Perfusion fixation at physiologic intraocular pressure caused a 47% decrease in the area of Schlemm's canal and a mean increase of 13.4% in the relative amount of empty space in the juxtacanalicular tissue compared with immersion-fixed fellow eyes. Perfusion of fixative did not appear to cause washout of extracellular material. Perfusion-fixed tissue appears preferable for studies of Schlemm's canal and for ultrastructural studies of the aqueous outflow pathways within the juxtacanalicular tissue.

Aged↗
PubMedDate not supplied

mRNA in-situ hybridization using biotinylated oligonucleotide probes: implications for the diagnostic laboratory.

It is now possible to detect low copy numbers of messenger ribonucleic acid (mRNA) while retaining good histologic morphology for the determination of specific gene expression in diseased tissues. This technology will allow the pathologist to provide important prognostic information about tumors (expression of oncogenes and growth factors), to identify the subclones within the tumor which may be most likely to metastasize (expression of adhesion molecules and proteases) and to identify etiologic genetic aberrations (viral insertions). A technique for in-situ hybridization to mRNA has been developed for use in formalin fixed paraffin embedded tissues which is suitable for a hospital histology laboratory. Optimal conditions for the procedure were determined by using a biotinylated poly (d)T oligonucleotide probe. Results were dependent on the tissue type, fixation time, condition of the tissue prior to fixation, and degree of digestion before hybridization. The temperature and conditions of hybridization were optimized so that the poly d(T) control probe and the longer test probe could be run simultaneously. Streptavidin and avidin alkaline phosphatase detection systems were tested using levamisole to minimize background staining, and a biotin blocking agent to reduce reaction to renal tubular biotin. Increasing the temperature of stringency washes did not significantly improve the specificity but had a markedly detrimental effect on tissue morphology. The mRNA appears to remain stable within routinely fixed surgical material over long periods of time allowing for large retrospective studies. A review of c-erbB-2 expression in 16 human breast lesions was carried out comparing mRNA in-situ hybridization to immunoperoxidase and cytosolic methods. By direct localization of both message and antigen, it was possible to demonstrate focal positivity that cytosolic methods did not detect. Aberrant translation was noted in one case, and c-erbB-2 expression in non-malignant breast was detected in two cases.

Animals↗
PubMed2006

Antigen-epitope retrieval to facilitate proteomic analysis of formalin-fixed archival brain tissue.

Formalin is a routine fixative facilitating tissue preservation and histopathology. Proteomic techniques require freshly frozen specimens, which are often difficult to procure, and methods facilitating proteomic analysis of archival formalin-fixed brain tissue are lacking. We employed antigen-epitope-retrieval principles to facilitate proteomic analysis of brain tissue that had been fixed and stored in formalin for 3-7 years. Twenty-micrometer-thick cryopreserved OCT-embedded sections from inferior temporal cortex of human (7 years in formalin) or mouse brain specimens (3 years in formalin) were hematoxylin-/eosin-stained. Approximately 16-64-mm2 areas of the tissue sections were manually scraped off slides, or approximately 2 mm2 of human brain cortex was captured off membrane-coated slides using laser microdissection. Tissue was treated using various pH and temperature conditions prior to trypsin digestion and nano-LC-MS/MS. The largest number of proteins were retrieved by solubilization at pH 9 at 95 degrees C for 1 h; treatments at pH 4 or 6 at 25 or 65 degrees C were generally ineffective. Three-year formalin-fixed murine tissue did not yield more proteins compared to human tissue. Use of formalin-fixed tissue for proteomics is an invaluable tool for medical research. The combination of proteomics and microdissection enables selective enrichment and identification of novel, unique, or abundant proteins that may be important in pathogenesis.

Animals↗
PubMed1975

[Osteosynthesis in the presence of soft-tissue lesions: delayed internal fixation and indications for conservative treatment (author's transl)].

When there are concomitant soft-tissue lesions with closed fractures, delayed osteosynthesis is indicated to prevent infection. Nonetheless, primary osteosynthesis is rarely necessary. In the case of open fractures, internal fixation should only be performed when bone and the materials used for fixation can be covered with tissues that are well supplied with blood. In case of doubt the surgeon can rely on the fact that most fractures can be effectively treated by conservative methods, and it is wise in such cases to forego the advantages of osteosynthesis and avoid the risks involved.

Adolescent↗
PubMed2004

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Acetic Acid↗
PubMed2003

Ionic liquids in embalming and tissue preservation. Can traditional formalin-fixation be replaced safely?

Ionic liquids (ILs) can be used for embalming and tissue preservation. ILs does not cause tissue damage and the tissue colour remains unaltered after treatment. Microscopical morphology of tissues fixed in ILs is of better quality than that of tissues fixed in formalin. Tissue preservation depends on the type of ILs. Best results were obtained with 1-methyl-3-octyloxymethylimidazolium tetrafluoroborate, the density of which resembles that of water. The salt is nonvaporous and when used as a formalin substitute, it eliminates health hazards in the pathological laboratory.

Animals↗
PubMed1998

[Confocal laser scanning microscopy (CLSM) for validation of non-destructive histotomography of healthy bone tissue].

OBJECTIVES: Fixation (formalin), decalcification (sections) or mechanical treatment (grinding) all bear the risk of artifacts occurring during hard-tissue histology. Because studies on the etiology of pathological changes mostly focus on subclinical lesions, artifacts can simulate early changes or even be superimposed on existing changes. The objective of this study was to determine how artifacts can be reduced. MATERIAL AND METHODS: In confocal laser scanning microscopy (CLSM) a focused laser beam scans the surface of the specimens and penetrates into the tissue. The intensity of the remitted light is recorded. The confocal effect is due to an extremely small aperture (pin-hole), excluding light from out-of-focus planes of the sample. By stepwise movement of the object table, a tomographic series of tomographic images is obtained. Sound cortical bone samples of the lower jaw (n = 20) were studied by light microscopy and by CLSM, visualizing identical areas of a ground sectioned sample after H&E staining. Additionally, embedded and fresh blocks of tissue of the same bone sample were studied histotomographically in the CLSM. RESULTS: (1) Light microscopic micromorphology of cortical bone can be visualized adequately in the CLSM; (2) many structures that can be visualized by light microscopy only after special staining (e.g., osteozyte processes) can be visualized by the CLSM using sample blocks without pretreatment. CONCLUSION: (1) Nondestructive subsurface histotomography by CLSM totally excludes mechanical artifacts; (2) physicochemical artifacts can be handled more easily because fresh samples can be studied; (3) pseudo-three-dimensional imaging allows histological interpretation of the tissue that is equivalent to macroscopic tomographic techniques (CT, MRT).

Artifacts↗
PubMed1992

Scanning electron microscopic study on dendritic cells and fibroblasts in connective tissue.

Perfusion fixation and intravascular resin injection were used to study in situ the cells of superficial fascia (loose connective tissue) of the rat limb by scanning and transmission electron microscopy. These procedures enabled us to differentiate fibroblasts, representative cells of the connective tissue and so-called "dendritic cells," the other cellular constituent of the tissue, known as possible antigen presenting cells. "Dendritic cells" were amoeboid with some spatular processes by which the cells clung to the collagen fiber bundles. The fine structures characteristics of macrophages; they contained abundant primary and secondary lysosomes and expressed factor XIIIa in their cytoplasm. Fibroblasts, on the other hand, were attenuated, sheet-like cells whose edges were juxtaposed closely to collagen and elastic fibers, and were further anchored by microfibrils. The cells were usually interconnected to each other with gap junctions, thus producing a cellular network in the connective tissue.

Animals↗
PubMed1986

Studies on the actions of glutaraldehyde, formaldehyde, and mixtures of glutaraldehyde and formaldehyde on tissue proteins.

The effects of aldehyde fixation on tissue proteins was studied using SDS-polyacrylamide gel electrophoresis. Fixation with low concentrations of formaldehyde or glutaraldehyde had only a slight effect on the protein banding pattern. On the other hand, most of the original protein bands were absent after a short treatment with mixtures of formaldehyde and glutaraldehyde. Spectral absorption measurements showed a stronger absorption at both gamma = 280 nm and gamma = 235 nm with the glutaraldehyde-formaldehyde mixture than with glutaraldehyde alone.

Aldehydes↗
PubMed1986

An improved procedure for fixation and embedding of whole, intact lens tissue for light microscopy.

Fixation and embedding of whole, intact lens tissue are often difficult. During studies of morphologic changes in various experimentally induced cataracts, an improved procedure was developed, utilizing isomolar fixative and water-soluble embedding medium (glycol methacrylate), while avoiding dehydration with organic solvents. Different rat and human cataractous lenses were processed in toto using this method, and the results were uniformly good upon light microscopic evaluation. The potential of this fixation-embedding procedure for lens research is discussed.

Animals↗