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PubMed1990

[DNA extraction and Southern blot analysis in paraffin embedded material].

DNA was extracted from formaldehyde fixed and paraffin embedded tissue by the use of a modified extraction protocol. In all cases the recovered DNA was more degraded than DNA from fresh or frozen tissue. Fixation times of more than 4 days made it impossible to use the extracted DNA for Southern Blotting; DNA from paraffinized tissues not older than 1 or 2 years could be used for Southern Blotting after digestion with restriction enzymes, but we did not succeed in showing bands with restriction fragments more than 10 kb in length. We conclude that SBA analysis for malignant lymphomas with DNA extracted from routinely paraffin-embedded tissues isn't possible, because the restriction fragments looked for are in many cases longer than 10 kb.

Blotting, Southern↗
PubMed2005

Identification of L-selectin binding heparan sulfates attached to collagen type XVIII.

L-selectin is a C-type lectin expressed on leukocytes that is involved in both lymphocyte homing to the lymph node and leukocyte extravasation during inflammation. Known L-selectin ligands include sulfated Lewis-type carbohydrates, glycolipids, and proteoglycans. Previously, we have shown that in situ detection of different types of L-selectin ligands is highly dependent on the tissue fixation protocol used. Here we use this knowledge to specifically examine the expression of L-selectin binding proteoglycans in normal mouse tissues. We show that L-selectin binding chondroitin/dermatan sulfate proteoglycans are present in cartilage, whereas L-selectin binding heparan sulfate proteoglycans are present in spleen and kidney. Furthermore, we show that L-selectin only binds a subset of renal heparan sulfates, attached to a collagen type XVIII protein backbone and predominantly present in medullary tubular and vascular basement membranes. As L-selectin does not bind other renal heparan sulfate proteoglycans such as perlecan, agrin, and syndecan-4, and not all collagen type XVIII expressed in the kidney binds L-selectin, this indicates that there is a specific L-selectin binding domain on heparan sulfate glycosaminoglycan chains. Using an in vitro L-selectin binding assay, we studied the contribution of N-sulfation, O-sulfation, C5-epimerization, unsubstituted glucosamine residues, and chain length in L-selectin binding to heparan sulfate/heparin glycosaminoglycan chains. Based on our results and the accepted model of heparan sulfate domain organization, we propose a model for the interaction of L-selectin with heparan sulfate glycosaminoglycan chains. Interestingly, this opens the possibility of active regulation of L-selectin binding to heparan sulfate proteoglycans, e.g. under inflammatory conditions.

Animals↗
PubMed1987

A new approach to immunocytochemistry of 3',5'-cyclic guanosine monophosphate: preparation, specificity, and initial application of a new antiserum against formaldehyde-fixed 3',5'-cyclic guanosine monophosphate.

The development of a new 3',5'-cyclic guanosine monophosphate (cGMP) antiserum was initiated starting from the following considerations: (a) adequate fixation of cGMP is a prerequisite for a reliable demonstration of soluble cGMP, and (b) fixation might influence the specificity of the immunocytochemical demonstration of cGMP. Therefore, cGMP-protein conjugate was prepared in a way which equals tissue fixation. cGMP was coupled to bovine thyroglobulin using formaldehyde. Antibodies against this conjugate were raised in rabbits. The specificity of the antisera was evaluated in a gelatin model system. No immunoreactivity was observed with nucleotides other than cGMP or with rabbit preimmune sera. Immunoinhibition experiments showed that only the cGMP-formaldehyde-thyroglobulin conjugate and, to a lesser extent free cGMP, absorbed onto the antiserum. In rat brain an extensive localization of cGMP-immunostaining was found. Examples are hippocampus CAI and CAII, and cortical layers II and V. No cGMP-immunostaining was found in the cerebellum. In vitro incubated superior cervical ganglia showed cGMP-immunostaining in the large postganglionic neuronal cell bodies; this cGMP-immunostaining increased upon incubation of the ganglia in iso-osmolar 100 mM K+. In conclusion, we prepared a new-type highly specific antiserum against cGMP, suitable to demonstrate cGMP-immunoreactivity in tissue material.

Animals↗
PubMedDate not supplied

Method for anchoring biomechanical implants to muscle tendon and chest wall.

Reliable tissue fixation is of fundamental importance to the successful development of muscle powered motor prostheses. This report describes a series of canine implant trials used to develop stable tissue-device interface mechanisms. Muscle pumps were fitted with prototype tendon and chest wall anchoring schemes and secured to the ribs and humeral insertion of latissimus dorsi (LD) muscles. LD stimulation was initiated 1 week postimplantation and continued throughout the implant period to stress these fixation sites. Design modification and implant testing were continued until both muscle and chest wall attachment points were found to be stable. Chest wall fixation was best achieved using perforated metallic plates wired to the ribs, as opposed to bone screws or wire mesh, which were subject to degradation. Direct attachment of the native tendon by means of spiked clamping plates proved ineffective. Stable muscle attachment was ultimately achieved by replacing the humeral tendon with an artificial substitute formed from fine polyester fibers gathered into 6-8 bundles and sewn into the LD insertion. Braided into a single cord, these fibers were fixed to the device by means of spiked clamping plates. Based on these findings, we conclude that perforated anchor plates and multifibrous artificial tendons can function as effective tissue-device interface mechanisms.

Animals↗
PubMed2002

Immunoglobulin heavy chain gene analysis in lymphomas: a multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays.

Determination of monoclonality through an evaluation of immunoglobulin heavy chain (IgH) gene rearrangements is a commonly performed and useful diagnostic assay. Many laboratories that perform this assay do so by the polymerase chain reaction (PCR). To evaluate current methods for performing IgH gene testing, 19 different Association of Molecular Pathology (AMP) member laboratories analyzed 29 blinded B cell and T cell lymphoid neoplasm samples of extracted DNA and formalin-fixed, paraffin-embedded (FFPE) tissue and were asked to complete a technical questionnaire. From this study, it is clear that Southern blot analysis remains the diagnostic gold standard, with a 100% diagnostic sensitivity and specificity. There was, however, remarkable heterogeneity in the performance of, and results obtained from, IgH PCR assays with diagnostic sensitivity ranging from over 90% to as low as 20%, when evaluating the same specimens. Many laboratories overestimate the diagnostic sensitivity of their IgH PCR assay, and there was a significant, and under appreciated, drop-off (from 61.3% to 41.8%) in detection in paired FFPE as compared with fresh/frozen tissues. Fixation has a dramatic impact on the inability to perform the test on FFPE (43.1%) versus DNA already extracted from fresh or frozen tissue (2.8%). A number of variables that affected the outcome of IgH PCR were identified. Strategies that improved the detection of monoclonal IgH rearrangements include: the addition of FRII to the FRIII upstream primer (increasing detection from 57.3% to 73.6%) and the use of the FR3A rather than the FR3 FRIII primer (increasing detection from 54.7% to 69.7%). Although numerous variables (from DNA extraction to PCR product detection) were evaluated, making it difficult to mandate alterations in laboratory practice, these findings ought to prompt diagnostic molecular pathology laboratories to reevaluate their claims of sensitivity, as well as their methodologies. Both pathologists and surgeons need to ensure that not all submitted material is fixed, if there is adequate sample. Importantly, there is a need for greater standardization to reduce the unacceptably high false negative rate of this crucial diagnostic assay.

Blotting, Southern↗
PubMed2005

L-Arginine does not affect renal morphology and cell survival in ischemic acute renal failure in rats.

BACKGROUND: L-Arginine (L-Arg), a substrate of nitric oxide synthases, improves renal function in ischemic acute renal failure (iARF). We evaluated whether L-Arg improves renal morphology and cell survival in the course of iARF. METHODS AND RESULTS: iARF was induced in rats by bilateral clamping of renal arteries for 45 min. L-Arg was applied intraperitoneally during clamping, and orally during 14 days of follow-up. Morphology and cell survival of renal cortical and medullar tissue was analyzed on days 1, 3, 7, and 14 of follow-up, using toluidine blue staining and immunohistochemistry of perfusion-fixated tissue, and Western blot analysis of tissue homogenate. Renal tubular injury showed typical features of necrosis and was most severe on days 1 and 3 after clamping, predominantly in S3 segments, with almost complete recovery by day 14. Enhanced medullar monocyte infiltration, determined by ED-1 expression as well as by immunohistochemistry, and enhanced expression of proliferating cell nuclear antigen (PCNA), indicative of proliferation and regeneration, accompanied these morphological changes. Compared to controls, L-Arg had no impact on renal morphology, ED-1, and PCNA expression. Furthermore, expression of markers of apoptosis Bcl-2, Bax, and cleaved caspase-3 was only slightly increased in iARF rats, compared to sham-operated animals, and was also not influenced by L-Arg. CONCLUSION: Despite its repeatedly reported positive impact on renal function as also shown in our model, L-Arg does not alter cell death and proliferation in the course of iARF in our model. Thus, different mechanisms have to be considered, in particular improved intrarenal hemodynamics.

Acute Kidney Injury↗
PubMed2002

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification.

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Animals↗
PubMed2003

Secure flap fixation to bony tissue using an anchoring system in skull-base surgery.

The reconstruction of extensive skull-base defects using flap transfer decreases crucial postoperative complications. However, flap fixation to bony tissue is often difficult, especially if there is no soft tissue to pass sutures through, and unsteady flap fixation causes dead space formation or displacement of the transferred flap due to gravity. The authors used an anchoring system for secure flap fixation in seven cases with difficult flap fixation, using the conventional suturing technique. Anchors were inserted at strategic points in the bony tissue, and secure flap fixation was achieved in all cases. Postoperative CT scans showed no dead space formation, and major postoperative complications were not observed. The anchoring system can be buried in bony tissue with a thickness of more than 4 mm, and it enables reliable flap fixation and dead space obliteration. Although the cost is relatively high, the procedure is simple and lowers the risk of crucial postoperative complications in skull-base surgery.

Adolescent↗
PubMed1995

Tau immunoreactivity associated with aluminum maltolate-induced neurofibrillary degeneration in rabbits.

Intracisternal administration of aluminum maltolate to rabbits produces a marked argyrophilic neurofibrillary degeneration (NFD) which is also immunoreactive for both phosphorylated and non-phosphorylated microtubule associated protein tau. Using tissue fixation in PBF, the monoclonal antibodies Tau-2 and AT8 stain the NFD. Dephosphorylation markedly reduces the positivity of AT8. Using PLP-fixed tissue, monoclonal antibody Tau-1 also immunostains aluminum-induced NFD.

Aluminum↗
PubMed1991

Redistribution of calbindin-D28k in chick intestine in response to calcium transport.

Vitamin D and its hormonally active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are known to alter several parameters associated with stimulated intestinal Ca2+ transport: levels of calbindin-D28K, tubulin, and endosomal-lysosomal organelles containing Ca2+, and calbindin-D28K. In the present study the as yet unexamined relationship among Ca2+ transport, calbindin-D28K, and microtubules was studied by immunofluorescence microscopy. In vitamin D3-treated or 1,25-(OH)2D3-treated chicks, in the absence of Ca2+ transport, immunofluorescence microscopy of intestinal tissue fixed at 25 C indicated a colocalization of calbindin-D28K and tubulin along epithelial cell brush border and basal-lateral membranes. Initiation of in situ Ca2+ absorption for 10, 20, or 30 min before tissue fixation resulted first in increased punctate calbindin-D28K staining and then in a progressive decrease in intestinal cell- and microtubule-associated calbindin-D28K, with a concomitant increase in calbindin-D28K labeling in the villus core. When intestinal tissue from 1,25-(OH)2D3-treated chicks was chilled to 4 C before fixation (a procedure shown by others to cause microtubule depolymerization), evaluation by immunofluorescence microscopy revealed diffuse cytoplasmic staining of both the immunoreactive tubulin and its associated calbindin-D28K. These results indicate the possible involvement of calbindin-D28K with tubulin during the process of Ca2+ transport and the secretion of the calbindin-D28K as a consequence of the overall transport process. Electron microscopy with immunogold labeling revealed intestinal epithelial calbindin-D28K to be localized inside of small vesicles and lysosome-like structures, with sparse cytoplasmic labeling. Subsequent electron microscopic analysis of intestinal epithelial microtubules prepared by polymerization and depolymerization revealed immunogold labeling in coprecipitated vesicular remnants, with consistently light staining of filaments traversing segments of the microtubules. In biochemical studies, isolation of intestinal microtubules or tubulin by three distinct procedures revealed increasing levels of associated calbindin-D28K as a function of time after 1,25-(OH)2D3 repletion of vitamin D-deficient chicks. Addition of calbindin-D28K to intestinal microtubules isolated from vitamin D-deficient chicks exhibited saturable binding when exogenous calbindin-D28K reached levels comparable to those present in vitamin D-replete chick intestine. Collectively, these results suggest that calbindin-D28K is predominantly located in membrane-delimited vesicles, with a very minor component associated with filamentous elements that can be isolated with tubulin and microtubules. Additionally, calbindin-D28K is dynamically involved in Ca2+ transport in the intestine.

Animals↗
PubMed1986

Observations on the effect of movement on bone ingrowth into porous-surfaced implants.

Although porous-surfaced orthopedic implants have been designed for fixation by bone ingrowth, there is clinical evidence that this does not always occur. Initial implant movement relative to host bone can result in attachment by a nonmineralized fibrous connective tissue layer. The ranges of movement that result in either bone or fibrous connective tissue fixation are observed in dogs in two independent studies. Experimentally, bone ingrowth can occur in the presence of some movement, albeit very small (up to 28 mu), while excess movement (150 mu or more) can result in attachment by mature connective tissue ingrowth.

Animals↗
PubMed1996

Autologous pericranial graft resurfacing after high condylectomy and discectomy of the temporomandibular joint in rabbits.

PURPOSE: This study tested the effects of an autologous pericranial graft placed over the condyle on healing after high condylectomy and discectomy. MATERIALS AND METHODS: Sixteen young adult New Zealand white rabbits were divided into four test groups and a control group. Three animals from the test group and one control animal were killed at 2, 4, 8, and 12 weeks postoperatively. Each experimental animal received a high condylotomy and discectomy bilaterally. In addition, on one side a pericranial graft was placed on the shaved bone and immobilized with a tissue adhesive. RESULTS: The results showed better healing of the articular defects in the joints where grafts had been placed, including earlier and more complete soft tissue covering, more substantial neochondrogenesis, and better organization of the regenerated articular cartilage. CONCLUSION: Pericranial grafts may be helpful in facilitating healing after high condylectomy. However, technical improvements in the method of tissue fixation may be necessary.

Animals↗
PubMed1958

Polarization optical properties of the pancreatic acinar cell of the mouse.

The birefringence of fresh and fixed mouse pancreatic acinar tissue was studied, utilizing whole mounts of pancreas from which the mesentery had been removed. Fresh pancreas in Tyrode's solution demonstrated positive birefringence with respect to the radial axis (the axis radiating from the nucleus as spokes from a wheel). Formol fixation reversed the sign of birefringence to negative with respect to the radius. The magnitude of birefringence increased with longer fixation. Neutral formol also reversed the sign to radially negative, but the magnitude did not increase with longer fixation. Tissue fixed in 2 per cent osmium tetroxide or potassium permanganate demonstrated strongly negative birefringence with respect to the radius. The cytoplasm of tissue fixed in acetic acid, though finely granular, still possessed radially positive birefringence. Birefringent areas could be seen in tissue fixed in potassium dichromate, but the sign of birefringence could not be determined. Ethyl alcohol, chromic acid, picric acid, and mercuric chloride all produced a brilliant cytoplasm in which no birefringence could be demonstrated. Freezing markedly decreased the radially positive birefringence of fresh tissue. Fresh tissue placed in increasing concentrations of glycerol demonstrated increasing radially positive birefringence. When formol-fixed tissue was placed in glycerol, the radially negative birefringence decreased. Osmium tetroxide-fixed tissue in 50 per cent glycerol was isotropic. The granularity of ethyl alcohol-fixed tissue disappeared in 50 per cent glycerol, and radially positive birefringence was evident. Frozen tissue showed increasing radially positive birefringence in increasing concentrations of glycerol. The results are discussed in relation to theories of fixation.

Acinar Cells↗
PubMedDate not supplied

The case for perfusion fixation of large tissue samples for ultrastructural pathology.

We present the case for the perfusion fixation of large, freshly isolated tissue samples of liver and lung from dog, rat, and mouse. Individual lobes of liver and lung were fixed by vascular perfusion using a technique that is simple and quick to perform and results in a reproducibly high standard of ultrastructural preservation compared to immersion fixation. A major advantage to the use of this technique lies in the ability to provide tissue samples for diagnostic ultrastructural pathology and toxicological pathology while avoiding the need for whole-organ or whole-body perfusion fixation. This advantage therefore permits the use of fresh tissue from the same organ for other investigative purposes (e.g., drug metabolism studies and pharmacokinetics), thereby allowing correlation of structure and function. The advantages of perfusion fixation compared to immersion fixation are discussed, and potential applications for tissue preparation of postmortem specimens and also for scanning electron microscopy are indicated.

Animals↗
PubMed2007

Experimental validation of noninvasive referencing in navigated procedures on long bones.

Navigation procedures in orthopedic surgery require fixation of reference markers to the anatomic region of interest. Inadequate fixation might lead to micromotion or loosening of the reference marker, consequently causing registration failures or errors in navigation. Osseous rigid fixation is usually achieved by minimally invasive Schanz screws or pins. The goal of this study was to evaluate a non invasive external fixation device, a headband so far used in cranial navigation, as an alternative invasive fixation technique to reference markers in the femur. A common navigation system with an adapted trauma software application was used to track the positions of the soft tissue-attached headband relative to an invasive reference marker on the femur during manipulations of the thigh. Relative translative and rotational movements of the headband were measured during defined movements of the hip and knee and manipulations of the headband itself. The results revealed high translative and rotational movements, up to 6 mm and 3 degrees , respectively, due to minor manipulations of the affected lower extremity. Noninvasive soft tissue fixation with a headband does not allow rigid fixation for accurate navigated registration or operative procedures at the femur. Necessary intraoperative movements or manipulations would cause substantial registration failures. Invasive fixation techniques with screws or pins are still the method of choice.

Bone Screws↗
PubMed1983

Monoclonal antibody (F5) to human prostate antigen.

Hybridoma culture F5 has been developed which secretes monoclonal antibody (McAb) directed to an epitope of a prostatic glycoprotein of Mr 34 kD (Prostate Antigen, PA). Tissue levels of PA have been evaluated using a competitive-binding enzyme-immunoassay based upon the inhibition of McAb binding activity to purified antigen. Results indicated the specific occurrence of high antigen concentrations in extracts prepared from prostatic tissues. The antigenicity of epitope F5 is resistant to tissue fixation and embedding protocols, and has been demonstrated upon immunoperoxidase staining procedures. Immunoperoxidase data strongly indicate that McAb F5 possesses a singular specificity towards prostatic epithelial cells. Other tissues, whether normal or cancerous, fail to express this determinant. Specimens examined included epithelial and nonepithelial tissues along with a panel of carcinomas and sarcomas. The antibody was able to detect tumor cells at extra-prostatic sites and represents a powerful probe for the detection and differential diagnosis of metastatic cancer of the prostate.

Animals↗