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[Effect of fixation in Carnoy's solution on nucleic acid and protein concentration in the rabbit superior cervical sympathetic ganglion].

By means of biochemical techniques, the stability of RNA, DNA and protein contents has been demonstrated in the rabbit superior cervical sympathetic node fixed in Carnoy's solution for 2 hours at 4 degrees C. A 2 hour fixation in Carnoy's fluid at room temperature, results, however, in a considerable loss of RNA (21%). The loss of DNA and protein did not exceed 2% of the total amount of the fresh ganglion tissue. Fixation at a higher temperature (37%) increased the loss percentage protein and of nucleic acids: 3.1, 5.5 and 42%, for protein, DNA and RNA, resp.

Animals↗

Preparation technique and new contrast medium for radiography of biologic specimens.

An easily prepared contrast medium for radiography of vascular and ventricular systems is described. The accompanying fixation methods are discussed. Utilizing a few, readily available components and chemicals, technologists should be able to easily prepare a contrast medium that will produce, both before and after preservative tissue fixation, a quality specimen for radiography. The methods are easily applicable to most biological specimens.

Contrast Media↗

The Mitek Mini GII anchor introduced for tendon reinsertion in the hand.

The Mitek Mini GII anchor is showcased as a relatively new device that provides a rapid, simple method for bony reattachment; here, it is introduced for the fixation of flexor or extensor tendons at their insertion. Transosseous tunnels with limited visualization, pull-out sutures, and external devices can now be avoided if desired. Applications for soft-tissue fixation should in no way be limited to the small bones of the hand. Expense of the apparatus remains its only major drawback.

Adolescent↗

Morphological changes induced by heterologous antibodies in the rat liver.

The dynamics of organ fixation of heterologous antibodies were studied with the immunofluorescent technique. This allowed to conclude to the presence of liver-specific antibodies in the immune serum on the basis of the tissue fixation of antibodies. A perivascular round cell infiltration is characteristic of the liver damage induced by antibodies. Dilatation of the rough endoplasmic cisterns and a condensation of the matrix was revealed by electron microscopic pictures. It is concluded on the basis of the regressive phenomena and of the mesenchymal reactions that some importance can be attached to humoral immunity in immunopathological processes.

Animals↗

Retrospective assessment of liver cell proliferation via PCNA: a comparison with tritiated thymidine.

Cell proliferation (S phase response) in archival liver tissues of partially hepatectomized rats was determined via proliferating cell nuclear antigen (PCNA) immunohistochemistry. These results were compared with the S phase response assessed previously in the same tissues via tritiated thymidine (Tdr) autoradiography. The effect of prolonged tissue fixation on PCNA immunohistochemistry was compared in two studies: study A, the liver was fixed for a maximum of 7 days and then embedded in paraffin and stored for approximately 18 months, while in study B, the liver was fixed in formalin for 7 years and then embedded in paraffin and stored for approximately 18 months until sectioning and immunostaining. PCNA immunostaining was successful in the liver sections of both studies, irrespective of the length of formalin fixation. Furthermore, the S phase labeling indices (LI) determined via PCNA and Tdr were comparable, although not identical, in the two studies. Therefore, use of PCNA immunohistochemistry should allow retrospective staining of rodent tissues for the assessment of cell proliferative activity in formalin-fixed organs from previously conducted toxicity and carcinogenicity studies.

Animals↗

Kupffer cells and PIMs in acute experimental African swine fever.

An ultrastructural study of Kupffer cells and pulmonary intravascular macrophages (PIMs) of healthy and African Swine Fever (ASF)-infected pigs was carried out. A vascular perfusion method was performed in order to obtain an optimal intravascular morphology and tissue fixation. The infection developed acute ASF lesions in both organs. Both Kupffer cells and PIMs were studied at different stages of infection. The differences observed in both macrophagic cells from uninfected and infected tissues are shown and discussed.

African Swine Fever↗

Microwave cell death: Enzyme histochemical evaluation for metastatic carcinoma of the liver.

We have reported that microwave cell death is a unique cell death preserving not only cell and nuclear shapes but also immunohistochemical antigenicity. However, their enzyme activity was lost, which indicated cell dysfunction and death. This peculiar observation implies that the microwave effect is likely an 'in situ' tissue fixation and that this type of cell death is morphologically different from cell death, by either oncosis or apoptosis, as previously known. To confirm whether this peculiar cell death was observed also in human tissue samples, we examined clinical samples from patients with metastatic liver cancer, which were treated with microwave irradiation. They were examined immunohistochemically for human Ki-67 antigen and proliferating cell nuclear antigen and enzyme histochemically for alkaline phosphatase, and the same morphological changes that were observed in microwave-treated rat liver were found. In conclusion, we believe that routine hematoxylin-eosin stain alone is not a suitable method to evaluate microwave treatment for cancer because microwave coagulation therapy-treated cells preserved their nuclei and cellular architectures, even after 3 months. For microwave-treated tumors, enzyme histochemistry is helpful to determine its effectiveness.

Aged↗

The distribution of retinol-binding protein and its mRNA in the rat eye.

Although a constant supply of retinol is a critical requirement for the visual cycle, the molecular mechanisms underlying retinol delivery, uptake, storage, and transport in the eye are not well understood. Previously the synthesis of serum retinol-binding protein (RBP) in the mammalian eye was reported. Now the distribution of RBP and RBP mRNA in the rat eye has been studied by immunohistochemical and in situ hybridization techniques has been studied. The RBP mRNA was present only in the cytoplasm of retinal pigment epithelial (RPE) cells, terminating abruptly at the pars plana. On the other hand, RBP immunoreactivity was more widespread. The most intense immunostaining was present in retinal ganglion cells, the corneal endothelium, and under certain conditions of tissue fixation, the corneal epithelium. Consistent but less intense immunoreactivity was detected in the photoreceptors, Müller cells, inner plexiform layer, ciliary epithelium and stroma, iris epithelium, retinal pigment epithelium, lacrimal glandular epithelium, and periorbital soft tissues. These findings suggest that RBP synthesized by the RPE may be secreted to various ocular locations. However, at present, uptake from plasma cannot be excluded as another possible source of ocular RBP. In the plasma, holo-RBP (the retinol-RBP complex) is transported in complex with another plasma protein, transthyretin (TTR). This substance is also synthesized by the RPE and its distribution in the eye is similar to that described for RBP. Taken together, these findings support the proposal that ocular RBP and TTR may function cooperatively in the intraocular translocation of retinol.

Animals↗

Microfilaments and the hydrosmotic action of vasopressin in toad urinary bladder.

The role of cytoskeletal elements in the antidiuretic hormone (ADH)-induced hydrosmotic response of the toad bladder has been investigated with the microfilament and microtubule disrupting agents, cytochalasin B and colchicine. In the presence of a transmural osmotic gradient, addition of ADH resulted in a measured osmotic permeability coefficient (Pf) of 190 +/- 12 micrometers/s; preincubation with cytochalasin B (2 x 10(-5) M) reduced this value to 138 +/- 6. However, ADH-induced diffusional permeability to tritiated water (PD) was enhanced by 17% with cytochalasin B. The toxin caused a pronounced vacuolation in granular cells only when both ADH and an osmotic gradient were present. This led to experiments with brief glutaraldehyde fixation after hormonal stimulation but before osmotic gradient imposition. Subsequent osmotic flow measurements yielded Pf values of 180 +/- 18 and 210 +/- 7 micrometers/s for ADH and ADH + cytochalasin B, respectively. With gradient imposition only after tissue fixation, no structural alterations were found. We conclude that cytochalasin B inhibition is through disruption of the intracellular portion of the pathway for osmotic flows. Comparable experimentation with colchicine indicated that its interference is primarily with ADH action on the mucosal membrane itself. These results may bear on the significance of particle aggregation in the mucosal membrane, which has been thought to indicate ADH-induced water channels but which is reported to be precluded by cytochalasin B treatment.

Animals↗

Soft tissue reconstruction in the shoulder. Comparison of suture anchors, absorbable staples, and absorbable tacks.

We evaluated three mechanical soft tissue fixation devices (SuperAnchor, Suretac, and the Instrument Makar [IM] Bioabsorbable Staple) in a cadaveric model by examining ultimate tensile failure and modes of failure in simulated Bankart repairs. We attempted to realistically evaluate the strengths of soft tissue reattachment procedures at the anterior glenoid under worst-case conditions--load to failure. Twenty fresh-frozen cadaveric shoulders were used in this investigation. Each of the three techniques was performed in each anterior glenoid rim at one of three locations: superior, middle, or inferior. The subscapularis muscle-tendon was harvested, used in the repair, and loaded to failure. The mean load at failure for the SuperAnchor was 217.32 N; for the IM Staple, 132.32 N; and for the Suretac, 122.37 N. A two-sample t-test demonstrated that the load at failure for the SuperAnchor was statistically greater (P < 0.001) when compared with the IM Staple and Suretac. There was no statistical difference between load at failure for the Suretac and the IM Staple. The most common failure mode for the Mitek was suture breakage (71%). Anchor pullout from bone was the most common failure mode for the IM Staple (75%) and Suretac (94%).

Analysis of Variance↗

Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval.

The influence of antigen retrieval (AR) technique on immunoreactivity in formalin-fixed, paraffin-embedded tissues is recognized. In focal reactive overgrowths of oral mucosa, we noted that patterns of immunoreactivity to a fibronectin polyclonal antibody were dependent on methods of AR. To establish these patterns we investigated eight pyogenic granulomas and eight fibroepithelial polyps. In the absence of AR no immunoreactivity was observed. After alpha-chymotrypsin AR a band of intense immunoreactivity was associated with vascular endothelial cells, with either minimal or no staining of connective tissue. After microwave AR immunoreactivity was observed in connective tissue, especially in the subepithelial region, but there was no specific vascular staining. After autoclave AR immunoreactivity was observed in connective tissue and also in epithelial nuclei. To determine if these results represented either changes induced by tissue fixation and processing or exposure of epitopes normally masked in vivo, we investigated frozen sections from two fibroepithelial polyps and one pyogenic granuloma. In frozen sections immunoreactivity was observed around vascular endothelial cells and within connective tissue, especially in the subepithelial region, but not in epithelia. This study provides indirect evidence that alpha-chymotrypsin and heat-mediated AR protocols expose different masked epitopes, and reinforces the need to undertake appropriate pilot studies for immunohistochemical investigation of archival tissue.

Antibodies↗

Ultrastructure of the fibrous tissue surrounding internal fixation devices.

The possible bursal nature of the fibrous tissue that develops like a capsule over internal fixation devices was studied ultrastructurally in 13 human and four canine cases. In both species, the basic structural pattern was similar. The capsule had two distinct zones. The superficial compact zone appeared as a pseudomembrane, and consisted of elongated fibrocytes arranged transversely in a collagenous and microfilamentous matrix. In the deep zone, the cytoplasm and its constituents of connective tissue cells varied from meager to abundant. The structural organization of the capsule as well as its cellular characteristics did not conform to the specificities of an adventitious bursa. The capsular tissue did not appear to be affected by such host factors as age; implant alloy composition; the type of the underlying bone or its abnormalities; duration of implantation, or superficial or deep location of the metal device. Inflammation rarely occurred in the capsular soft tissue, including those cases where pain developed over the implants following an uneventful bone union. Because metal implants do not induce formation of adventitious bursa, pain over an uncomplicated implant is not likely to be due to a bursitis.

Adipose Tissue↗

Improved method for post-embedding cytochemistry using reduced osmium and LR white resin.

We established an improved method for post-embedding cytochemistry by which highly specific cytochemical reactions on excellent cellular ultrastructures are possible. The method is a combination of post-fixation in potassium ferrocyanide-reduced OsO4 and embedment in acrylic-based LR White resin. It permits both immuno- and lectin-gold cytochemistry with fine ultrastructures comparable to those obtained by conventionally osmicated and epoxy-embedded tissues. Fixation with reduced osmium appeared to contribute to the preservation of immunoreactivity and membranous structures. By this method, the immunocytochemical localization of secretory proteins (amylase and chymotrypsinogen), actin filaments by polyclonal antibodies, 105 KD Golgi-associated protein by a monoclonal antibody (GF-1), and binding sites for gold-labeled lectin could be demonstrated. Also possible was multiple staining with enzyme cytochemistry of thiamine pyrophosphatase and immunocytochemistry of GF-1 and anti-amylase antibodies. This multiple staining made possible partial characterization of the trans-Golgi network in parotid acinar cells.

Acrylic Resins↗

Thymic T-cell lymphoma with the CD8+ (OX-8), CD4+ (W3/25) phenotype, induced in F344/NCr rats by nitroso-2-hydroxypropylurea.

Thymic T-cell lymphomas were induced by administration of nitroso-2-hydroxypropylurea (NHPU) at a dose of 1.6 mg by gavage twice weekly for each of 20 weeks in F344/NCr rats. Lymphomas began to appear 5-10 weeks after the first intubation. Primary and transplantable tumors were subjected to morphological, histogenetic and immunological characterization. Lymphomas composed of medium-sized lymphocytes (prolymphocytes) arose within individual thymic lobules, often in atrophic thymic lobes and metastasized slowly to the T-cell zones of the splenic white pulp. The tissue fixative was important for tumor morphology and classification, with Bouin's fluid markedly superior to formalin. Transplantable tumors were similar morphologically except for one lymphoblastic lymphoma, but all transplants metastasized quickly to spleen, liver, thymus, and other tissues. Fluorescence-activated cell sorter (FACS) analysis of primary and transplantable lymphomas revealed cells of the T-cell lineage, with tumor cells expressing both OX-8 (CD8) and W3/25 (CD4) antigens. Immunoperoxidase studies of spleen showed infiltration of OX-8+ tumor cells first into T-cell dependent areas of the splenic white pulp.

Animals↗

Visualization of interstitial cells of Cajal in living, intact tissues.

Interstitial cells of Cajal (ICC) appear to be a major element in pacing and signal transmission in the gastrointestinal tract. A prominent problem in the study of ICC has been the difficulty in observing them in intact tissues. We used several methods to visualize living ICC in freshly-dissected tissues: (1) Placing small crystals of the lipophilic dye DiI in the submucosal-circular muscle border in the mouse colon resulted in the labeling of living ICC-like cells. Two main morphological cell types, bipolar and multipolar, were noted. The DiI stain could be converted into a stable, electron-opaque product. Electron-microscopic observations showed that the labeled cells had the typical appearance of ICC reported in previous studies. (2) Living ICC in the region of the myenteric plexus (ICC-MP) in the small intestines of mice and guinea-pigs were observed with Nomarski optics. This enabled the visualization of ICC in living tissues, and the impalement of the cells with Lucifer yellow-filled microelectrodes. The dye-labeled cells had the morphological features of ICC-MP, and about 30% of them were found to be dye coupled to 1-21 other ICC. The identity of the cells as ICC was verified by electron-microscopy following photoconversion, and by c-kit immunohistochemistry. (3) Living ICC were labeled with a c-kit antibody that does not require tissue fixation. This resulted in the fluorescent staining of the entire ICC network. Single cells were labeled by dye injection, which provided a detailed picture of ICC morphology. This method was found to be suitable for a wide range of tissues. We expect that these three methods for identifying ICC in intact, living tissues will be useful for physiological and pharmacological investigations of ICC in a variety of gastrointestinal tissues.

Animals↗

[Permeability of cutaneous macrophage lysosomal membrane in delayed hypersensitivity to streptococcal antigens].

The acid phosphatase (APh) was studied in the skin macrophage lysosomes of animals with delayed-type hypersensitivity (DTH) to the antigens of group A streptococci or tuberculoproteins. The histochemical procedure for the APh determination was employed in the skin sections without any preliminary tissue fixation. Intradermal injection of specific antigens in DTH proved to lead to the increase of the lysosomal membrane permeability in the skin macrophages. These data support the supposition that the lysosome enzymes of macrophages can act as factors causing tissue destruction in DTH.

Acid Phosphatase↗

Detection of human papillomavirus in archival tissues. Comparison of in situ hybridization and polymerase chain reaction.

Formalin-fixed, paraffin-embedded tissues in pathology archives are an important resource for molecular epidemiology studies. Use of these tissues requires that assays be optimized to account for inevitable variations in tissue fixation and processing that occur in the performance of routine histology. We compared results of colorimetric in situ hybridization (ISH) to L1 consensus polymerase chain reaction (PCR) for detection and typing of human papillomavirus (HPV) in 180 blocks of archival tissues (up to 9 years in storage) from cervical cancer patients. Fifteen samples could not be amplified by PCR, but assays were concordant in 75.1% (124/165) of samples that could be analyzed by both methods. Similar numbers of ISH+/PCR- (23) and ISH-/PCR+ (18) cases were found. Eight of the 18 ISH-/PCR+ cases were attributable to PCR detection of HPV types not included in the ISH assay. This degree of concordance required individual optimization of assay conditions for each block. ISH and PCR assays for HPV yield complementary results, and both can be successfully applied to archival tissues.

Female↗

Soft tissue tendon graft fixation in serially dilated or extraction-drilled tibial tunnels: a porcine model study using high-resolution quantitative computerized tomography.

BACKGROUND: Tibial tunnel preparation may contribute to improved soft tissue graft fixation. HYPOTHESIS: Step dilation produces greater tunnel wall bone volume than does extraction drilling and increases fixation strength. Bioabsorbable interference screw divergence decreases fixation strength, regardless of tunnel preparation method. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty porcine tibias were divided into 2 groups of 10 with matching mean apparent bone mineral density. One group received 9-mm-diameter extraction-drilled tunnels, and the other group received 7-mm-diameter extraction-drilled tunnels followed by step dilation to 9 mm. High-resolution quantitative computerized tomography scans and voxel analysis techniques determined tunnel wall bone volume fraction. Screws secured 8.5-mm-diameter porcine grafts in the tunnels. Repeat scans were used to determine screw divergence. Cyclic loading was performed in a servohydraulic device before load to failure testing. RESULTS: The step dilation group had greater tunnel wall bone volume/total volume than did the extraction drilled group; however, a significant increase in fixation strength was not detected. Specimens with screw divergence angles less than 15 degrees had superior fixation and insertion torques compared with specimens with angles 15 degrees or more. Screw divergence correlated more strongly with fixation strength than did mean apparent bone mineral density or screw insertion torque. CONCLUSION: Step dilation increased tunnel wall bone volume/total volume, but fixation strength did not improve. Screw divergence >or=15 degrees decreases graft-bone tunnel fixation whether or not step dilation is performed. CLINICAL RELEVANCE: Screw alignment plays a greater role in anterior cruciate ligament graft fixation than does extraction drilling or step dilation tunnel preparation methods in healthy bone.

Absorbable Implants↗