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PubMed1995

Novel fixation of plant tissue, staining through paraffin with alcian blue and hematoxylin, and improved slide preparation.

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.

Alcian Blue↗
PubMed1989

The tension-stress effect on the genesis and growth of tissues. Part I. The influence of stability of fixation and soft-tissue preservation.

To evaluate the optimum conditions for osteogenesis during limb lengthening and to study the changes in soft tissues undergoing elongation, a series of experiments were performed on the canine tibia. The experiments used the transfixion-wire, Ilizarov circular external skeletal fixator in configurations of differing stability of fixation in combination with a second variable, i.e., preservation of the periosteum, bone marrow, and medullary blood supply. Both increased fixator stability, and maximum preservation of the periosseous and intraosseous soft tissues enhanced bone formation during limb lengthening. To assess the role that the direction of the elongation vector plays in osteogenesis, canine tibiae were widened rather than lengthened in a second series of experiments using an Ilizarov apparatus modified for lateral distraction. The new bone formed parallel to the tension vector even when perpendicular to the bone's mechanical axis. As in longitudinal lengthening, damage to the bone marrow inhibits osteogenesis occurring by the influence of a lateral tension-stress vector. In a third series of experiments, half- and full-circumference cortical defects were created in canine tibiae to study the osteogenic potential of the marrow. New bone formed rapidly, even when the marrow was separated from the surrounding periosseous soft tissues by a sheet of polyvinyl chloride, attesting to the importance of marrow element preservation during osteotomy for limb lengthening.

Animals↗
PubMed2003

The changes in crosslink contents in tissues after formalin fixation.

The aim of this study was to detect crosslinks of collagen and elastin in formalin-fixed tissue, to perform quantification of these crosslinks, and to investigate the effects of formalin fixation on crosslink contents in human yellow ligament and cartilage. Pyridinoline (Pyr) is a stable and nonreducible crosslink of collagen. Pentosidine (Pen) is a senescent crosslink formed between arginine and lysine in matrix proteins, including collagen. Desmosine (Des) and its isomer isodesmosine (Isodes) are crosslinks specifically found in elastin. It is useful to measure crosslink contents of collagen and elastin as a way of investigating the properties of various tissues or their pathological changes. If it is possible to evaluate crosslinks of collagen and elastin in formalin-fixed tissues, we can investigate crosslinks in a wide variety of tissues. We used HPLC to compare the concentrations of Pyr, Pen, Des, and Isodes in the formalin-fixed tissues with their concentrations in the frozen tissues. Pyr and Pen were detected in both the formalin-fixed yellow ligament and the cartilage, and their concentrations were not significantly affected by or related to the duration of formalin fixation. Des and Isodes were detected in the formalin-fixed yellow ligament but in significantly lower amounts compared to the frozen samples. We concluded that crosslinks of collagen were preserved in formalin, but crosslinks of elastin were not preserved in it. The reason for this might be that formalin did not fix elastin tissues sufficiently or it destroyed, masked, or altered elastin crosslinks.

Aged↗
PubMedDate not supplied

Hexazonium pararosaniline as a fixative for animal tissues.

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a "fixative" action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.

Albumins↗
PubMed1996

The effects of guided tissue regeneration and fixation technique on osseous wound healing in rabbit zygomatic arch osteotomies.

The development of fibrous nonunions after craniofacial surgery is thought to result from an interaction of biomechanical stress and the differential migration of various tissue types into the wound site during healing. The present study is designed to test this hypothesis through the manipulation of guided tissue regeneration and osteotomy fixation techniques in an experimental rabbit model. Bilateral, critical size (5 mm), vertical osteotomies (n = 32) were produced in the zygomatic arches of eight adult rabbits. The mobile bony segments were fixed rigidly or nonrigidly using bone microplates and screws or osteosynthetic wires. The defects were then covered with a resorbable collagen membrane or left uncovered. The rabbits were followed for 4 weeks with serial dorsoventral cephalographs and the zygomatic arches harvested for histological analysis. Cephalometric results revealed significantly (p < 0.001) increased bone growth in the margins of the defects covered with the collagen membrane; however, no significant (p > 0.05) differences were noted between fixation techniques. Histological analysis revealed that defects fixed rigidly and covered by the membrane showed the most rapid and organized osseous wound healing, followed by the defects that were fixed nonrigidly and membrane covered. The defects not covered by the collagen membrane showed invasion by fibroblasts resulting in fibrous nonunions. These results demonstrate the efficacy of guided tissue regeneration with a resorbable collagen membrane in preventing fibrous tissue ingrowth in large bony defects. The addition of rigid fixation at a potentially mobile site appeared to enhance bony trabecular organization but not the osteogenic rate in this rabbit model.

Animals↗
PubMedDate not supplied

The twist-lock concept of tissue transport and suture fixation without knots: observations along the Hong Kong skyline.

PURPOSE: To evaluate the load to failure and the mode of failure of a novel suture anchor construct that does not require knots (the "twist-lock" construct) and to compare it with a standard suture anchor construct (Corkscrew; Arthrex, Naples, FL). TYPE OF STUDY: Biomechanical single-pull load-to-failure study comparing the twist-lock construct to the Corkscrew suture anchor construct. METHODS: The twist-lock construct is a suture anchor system that does not use knots, instead using 3 consecutive twists between suture limbs to enhance internal interference between the suture limbs. This system maximizes internal interference by 2 mechanically verifiable friction-multiplier mechanisms: the cable friction effect and the wedge effect. After theoretically verifying the strength characteristics of the twist-lock system, the authors tested and compared its strength in vitro to that of a standard screw-type suture anchor system (Corkscrew). Unicellular polyurethane, which has been shown to accurately mimic the properties of cancellous bone, was used for implantation of suture anchors for the purpose of comparing the load to failure of 10 identical constructs in each of the 2 anchor systems. Axial single-pull loading to failure was performed with an Instron 5565 testing machine (Instron, Canton, MA). RESULTS: The average load to failure for the twist-lock group was 137.2 N, and the average for the Corkscrew group was 123.0 N, a difference of 14.2 N. This study shows that the twist-lock anchors failed at a load that was 12% higher than that of the Corkscrew group (P =.02). CONCLUSION: The twist-lock system is a suture anchor system that achieves suture fixation of soft tissue to bone without the need to tie knots. It shows single-pull loads to failure that are significantly higher than those of a standard suture anchor system.

Equipment Design↗
PubMed1994

Detection of proliferating cell nuclear antigen in paraffin-embedded specimens is dependent on preembedding tissue handling and fixation.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), a cell cycle-related protein used to estimate tumor growth fraction, is variable in formalin-fixed compared with methanol-fixed tissue specimens. This is assumed to result from conformational changes in the antigenic epitope induced by formaldehyde; therefore, to be susceptible to retrieval in archival specimens. In this study, formalin fixation reduced the intensity of staining and the number of positive cells to approximately 25% of those in methanol-fixed material. The washing of tissue specimens prior to methacarn fixation also reduced PCNA staining. Loss of staining was not restored after use of a commercial retrieval kit recommended for PCNA immunohistochemistry. Immunoblotting of formalin fixatives and saline washings after removal of tissue specimens consistently demonstrated the presence of PCNA-like activity in solution. We conclude that the exceptional solubility of PCNA is responsible for reduced immunostaining in formalin-fixed material, that the loss is irreversible, and that methanol or methacarn is the fixative of choice for PCNA immunohistochemistry.

Animals↗
PubMed1978

Use of nitrogen mustard N-oxide for the fixation of electron microscopic tissues.

Nitrogen mustard N-oxide was tried for the fixation of tissue for electron microscopy. A fixative consisting of 1% nitrogen mustard N-oxide, 1% glutaraldehyde and 1% paraformaldehyde buffered at pH 7.4 followed by 1% OsO4 buffered at pH 7.4 was found useful for the tissues examined: thyroid, anterior pituitary, adrenal gland and oviduct of mice. If the tissues are fixed and the sections are stained with uranyl acetate and lead acetate doubly, the follicle colloid, colloid droplets, and secretory granules containing thyroglobulin in the thyroid become higher in electron density. The cisterna of the maturing face of the Golgi apparatus, secretory granules, ribosomes, nucleolus and chromatin in the cells examined are extremely electron dense. Tubular elements of smooth endoplasmic reticulum in the adrenal cortical cell and microtubules in all the cells examined are also well preserved. The fixative containing nitrogen mustard N-oxide is useful also for cytochemistry. Using tissue fixed by this method and stained en bloc by uranyl acetate, the noradrenaline and adrenaline cells in the adrenal medulla are clearly distinguished by light microscopy.

Adrenal Medulla↗
PubMedDate not supplied

Blue-violet excited autofluorescence spectroscopy and imaging of normal and cancerous human bronchial tissue after formalin fixation.

Autofluorescence (AF) imaging is a powerful tool for the detection of (pre-)neoplastic lesions in the bronchi. Several endoscopic imaging systems exploit the spectral and intensity contrast of AF between healthy and (pre-)neoplastic bronchial tissues, yet, the mechanisms underlying these contrasts are poorly understood. In this report, the effect of formalin fixation on the human bronchi AF, hence on the contrast, was studied by spectrofluorometric point measurements and DAFE (Diagnostic AutoFluorescence Endoscopy) broad field imaging. Generally, formalin-fixed samples have higher AF intensity than in vivo, whereas the emission spectral shape is similar. Additionally, the spectrofluorometric data showed a moderate decrease of the AF intensity on (pre-)neoplastic lesions relative to the healthy bronchial samples. However, this decrease was lower than that reported from in vivo measurements. Neither spectral measurements nor imaging revealed spectral contrast between healthy bronchial tissue and (pre-)neoplastic lesions in formalin. These results indicate that epithelial thickening and blood supply in the adjacent lamina propria are likely to play a key role in the generation of the AF contrast in bronchial tissues. Our results show that the AF contrast in bronchial tissues was significantly affected by standard, 10% buffered, formalin fixation. Therefore, these samples are not suited to AF contrast studies.

Bronchi↗
PubMed1988

Evaluation of different fixatives and treatments for immunohistochemical demonstration of Coxiella burnetti in paraffin-embedded tissues.

Various fixatives and treatments such as acetone, methanol, Bouin fixative, modified Bouin fixative, 10% Formalin, modified methacarn, periodate-lysine-paraformaldehyde, acetone-methyl benzoate-xylene, and EDTA were evaluated for their effect on the immunoreactivity of Coxiella burnetii in paraffin-embedded tissues by using the avidin-biotin-peroxidase complex and the peroxidase-antiperoxidase procedure. C. burnetii antigen was shown to be present in liver, spleen, and uterus tissues of experimentally infected mice by all methods of fixation and treatment. A positive immunoreaction was seen in cytoplasmic vacuoles of macrophages, as extracellular rod-shaped organisms, and as residual particulate extra- and intracellular debris. Immunoreactivity and cellular preservation, however, varied substantially with the individual fixatives. Optimal immunostaining of C. burnetii was achieved by EDTA treatment and Bouin and acetone fixation. The avidin-biotin-peroxidase technique proved to be slightly more sensitive than the peroxidase-antiperoxidase procedure when primary antibody dilution was used as the criterion for sensitivity.

Animals↗
PubMed1991

Microwave energy fixation of plant tissue: an alternative approach that provides excellent preservation of ultrastructure and antigenicity.

Immunocytochemical techniques are confronted with the problem of obtaining adequate tissue preservation together with retention of protein antigenicity. Various methods, including freeze-drying and freeze-substitution, have been devised to circumvent this problem. In the present study, we report that microwave energy used in combination with low concentrations of glutaraldehyde (0.1%) and paraformaldehyde (2%) preserved the structural integrity of plant tissue and antigenicity of proteins. Tobacco leaf samples fixed in a time as brief as 15-20 s exhibited excellent preservation of fine structures. By contrast, specimens irradiated for shorter (5-10 s) or longer (30-40 s) periods showed poor morphological preservation. Microwave irradiation for 15-20 s was found useful for immobilizing large amounts of soluble antigens. The fast microwave fixation method was successfully used to preserve pathogenesis-related (PR) proteins, which were subsequently localized by a postembedding immunogold procedure. In addition to soluble antigens, cellulose subunits and pectic substances, two major plant cell wall components, were found to be highly preserved in microwave-irradiated tobacco plant tissue. The present study demonstrates that microwave fixation of plant tissue is a simple and inexpensive method that is easy to perform with commercially available microwave ovens. The incubation time for fixation is reduced from 2 h to 15-20 s without loss of fine structural details. This method will undoubtedly acquire increasing applicability and relevance in plant biology.

Antigens↗
PubMed1982

Evidence for cross-linking of cyclic AMP to constituents of brain tissue by aldehyde fixatives: potential utility in histochemical procedures.

The amount of cyclic AMP recovered from unfixed tissue sections of brain carried through immunohistochemical procedures was found to be small (less than 2 pmoles/mg protein) and constant despite wide variations in the amount accumulated prior to freezing and sectioning. However, treatment of brain slices with glutaraldehyde or formaldehyde in buffered sucrose solutions for one to two hours at 0 degrees rendered insoluble as much as 60% of the total accumulated cyclic AMP as judged by filtration of homogenates of treated tissue. Immunoreactive cyclic AMP was recovered from filters by extraction with warm, dilute acid. The fraction of filter-bound cyclic AMP was relatively constant over a wide range of initial tissue levels. The persistence of insoluble cyclic AMP was greatest in homogenates of slices treated with formaldehyde when maintained above pH 8.5; about 40% of this fraction was lost after two hours at 0 degrees. Incubation of tissue homogenates with formaldehyde and radioactive nucleoside or nucleotide derivatives of adenine, guanine, and cytosine resulted in a time-dependent appearance of insoluble radioactivity; compounds lacking an amino function were inactive. Pure proteins, including polylysine, also reacted with formaldehyde and 3H-cyclic AMP to produce radioactivity resistant to adsorption by charcoal. The reaction between cyclic AMP or adenosine, formaldehyde, and alkyl amines was examined using UV spectrometry. It is tentatively concluded that at 0 degrees formaldehyde is capable of rapidly producing a methylene-bridged adduct between primary alkylamines and the N6-amino function of purine nucleosides or nucleotides. Application of these results to the development of immunohistochemical procedures for the cellular localization of cyclic AMP will require the generation of antibody preparations with high reactivity for cyclic AMP derivatized at the N6-position.

Aldehydes↗
PubMed1991

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.

Acetone↗
PubMed1993

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

Animals↗