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At least 1,279 records · Page 71Linked to original sources
PubMed2003

Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus.

Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.

Acetic Acid↗
PubMed1996

Complex synaptic arrangements in the rat suprachiasmatic nucleus: a possible basis for the "Zeitgeber" and non-synaptic synchronization of neuronal activity.

A special type of complex synaptic arrangement occurs in the ventro-lateral portion of the rat suprachiasmatic nucleus. These arrangements are polycentric, with about equal numbers of pre- and postsynaptic elements. Because of an incomplete astroglial covering, these synaptic complexes are connected with each other and form a continuous reticulum or sponge-like system throughout the ventro-lateral region of the nucleus. In two partially reconstructed complex synaptic arrangements, boutons from retinal afferents could be seen to make up the majority of presynaptic elements. They form asymmetric and symmetric synaptic appositions with dendritic elements. Non-optic axo-dendritic synapses of unknown origin with asymmetric and symmetric appositions and dendro-dendritic synapses with symmetric appositions are also seen in complex synaptic arrangements. Within complex synaptic arrangements, dendrites often run in bundles, with some dendrites spiralling around others. Membranes of neighbouring dendrites are closely apposed. These interdendritic appositions are possibly ephapses and may, together with intersomatic contacts, mediate non-synaptic synchronization of neuronal activity in the suprachiasmatic nucleus, as described by other authors. The activity of optic and non-optic synapses in complex synaptic arrangements over a 24 h period may also produce an integrated response that influences the circadian rhythm of neuronal activity in this nucleus.

Animals↗
PubMed1995

Visualization of newt aragonitic otoconial matrices using transmission electron microscopy.

Otoconia are calcified protein matrices within the gravity-sensing organs of the vertebrate vestibular system. These protein matrices are thought to originate from the supporting or hair cells in the macula during development. Previous studies of mammalian calcitic, barrel-shaped otoconia revealed an organized protein matrix consisting of a thin peripheral layer, a well-defined organic core and a flocculent matrix inbetween. No studies have reported the microscopic organization of the aragonitic otoconial matrix, despite its protein characterization. Pote et al. (1993b) used densitometric methods and inferred that prismatic (aragonitic) otoconia have a peripheral protein distribution, compared to that described for the barrel-shaped, calcitic otoconia of birds, mammals, and the amphibian utricle. By using tannic acid as a negative stain, we observed three kinds of organic matrices in preparations of fixed, decalcified saccular otoconia from the adult newt: (1) fusiform shapes with a homogenous electron-dense matrix; (2) singular and multiple strands of matrix; and (3) more significantly, prismatic shapes outlined by a peripheral organic matrix. These prismatic shapes remain following removal of the gelatinous matrix, revealing an internal array of organic matter. We conclude that prismatic otoconia have a largely peripheral otoconial matrix, as inferred by densitometry.

Animals↗
PubMed2006

Optimizing conditions and avoiding pitfalls for prolonged axonal tracing with carbocyanine dyes in fixed rat spinal cords.

We have characterized a method of labeling of axons in the post-mortem spinal cord using a silastic disc holding pins coated with DiI and DiO at the rostral and caudal ends of the cord. We optimized the DiI and DiO tracing techniques under different conditions of fixative concentration (1% versus 4% paraformaldehyde, PF), at room temperature (RT) versus 37 degrees C for up to 24 weeks. Crystal coated pins embedded in a silastic disc provided a novel method of dye application. Confocal microscopy of longitudinal sections showed DiI and DiO labeled both the axonal membrane and myelin sheath. DiI diffused significantly longer distances than DiO. Both dyes migrated greater distances at 37 degrees C compared with RT. No significant difference of dye labeling was found between 1% and 4% PF fixation. After prolonged incubation there was evidence that dye diffused through the aqueous medium and produced circumferential labeling of the cord. Placing a wax seal around the labeling site prevented this non-contiguous labeling. Labeling of myelin sheaths at extended distances into the cord suggested that dye could migrate between cells with prolonged incubation periods. Our data suggested that higher temperature facilitated dye diffusion along the axons, and demonstrated that with caution DiI and DiO could be used as specific tracers in the same spinal cords.

Animals↗
PubMed1998

Cross-pin femoral fixation: a new technique for hamstring anterior cruciate ligament reconstruction of the knee.

A new method of femoral fixation for arthroscopic anterior cruciate ligament (ACL) reconstruction using semitendinosus and gracilis (ST/G) tendons is presented. Biomechanical evaluation of the method was performed with pullout tests using animal tissues simulating fixation of ST/G tendons passing around a 2.5-mm pin placed transversely through a femoral tunnel. Clinical assessment of 22 patients was performed according to IKDC, Tegner, Lysholm, isokinetic strength, and KT1000 standards. Fixation strengths ranged from 725 N to 1,600 N for yield and maximal loads to failure for 35-mm to 70-mm cross-pins. Clinical results at mean follow-up of 30 months showed one traumatic failure, IKDC 86% normal or nearly normal, Tegner 6 ave, Lysholm 93 ave, KT1000 86% < 3 mm side-to-side difference on maximal manual evaluation, and isokinetic strength testing of quadriceps 8% deficit with hamstrings 8% deficit. Cross-pin fixation of ST/G tendons may be one of the strongest modes of femoral graft attachment yet devised. Our clinical outcomes in this initial series compare well with previous reports of ACL reconstruction regardless of graft or technique.

Adolescent↗
PubMed1996

Paramyosin isoforms of Schistosoma mansoni are phosphorylated and localized in a large variety of muscle types.

Paramyosin, although a widely distributed muscle component among invertebrates, has hitherto not clearly been shown to occur in the muscles of schistosomes. Instead, it has been reported to occur in the tegument. In the present study, a specific antibody reacting with each of 10 isoforms of paramyosin was used for light microscopical immunolocalization in sections of Schistosoma mansoni. Specimens were fixed by a new method to immobilize antigens with uranyl acetate-trehalose-methanol. In cercariae, schistosomula, and adults, the circular and longitudinal muscles of the body wall, the dorsoventral muscles and those surrounding the gut and the pharynx as well as the fast moving cross-striated muscles of the tail of cercariae intensely reacted with the antibody. However, neither immunohistologically nor on Western blots of isolated tegument, were indications found for the presence of paramyosin in the tegument. In vivo phosphorylation and binding of anti-phospho-tyrosine and anti-phospho-serine antibodies show phosphorylation of paramyosin which probably is responsible for the generation of the isoforms.

Animals↗
PubMed2000

A novel, noninvasive imaging technique for intraoperative assessment of parathyroid glands: confocal reflectance microscopy.

BACKGROUND: Successful surgical management of primary hyperparathyroidism requires the ability to identify and distinguish normal from abnormal parathyroid tissue. Microscopic pathologic confirmation often helps with the diagnoses and decisions regarding the extent of parathyroid resection. Confocal reflectance microscopy (CRM) is an optical method of noninvasively imaging tissue without fixation, sectioning, and staining as in standard histopathology. The goal of this study was to determine if CRM imaging could be used to distinguish normal from diseased parathyroid tissue intraoperatively. METHODS: In this study, 44 parathyroid glands from 21 patients undergoing operations for primary hyperparathyroidism were imaged immediately after excision. CRM images were compared with conventional hematoxylin-and-eosin stained sections obtained from the same gland. The percentage area occupied by fat cells was calculated in images of both normal and diseased glands. RESULTS: Characteristic microscopic features of parathyroid glands were distinguishable by CRM and correlated well with histopathology. The stromal fat content of normal and diseased glands could easily be determined. The percentage area occupied by fat cells differed significantly (P <.00001) in normal glands (average, 23.0% +/- 10.9%) and adenomatous glands (average, 0.4% +/- 0.7%). CONCLUSIONS: CRM imaging rapidly revealed microscopic features that reliably differentiated normal and diseased parathyroid glands. The success of this preliminary ex vivo study promotes interest in further development of an in situ probe for in vivo clinical diagnostic use.

Adenoma↗
PubMed1996

Accordion-like structure of terminal hair follicles of the human scalp.

Terminal hair follicles extracted after ethylenediaminetetraacetic acid treatment preserved the basal cell surface of the outer root sheath well enough to examine the outer surface in detail. Light microscopic observations of whole mount specimens revealed several sawtooth-like structures of follicular epithelium in the lower portion of terminal hair follicle. Vertical sections of the human scalp also showed several invaginations of the outer root sheath of terminal hair follicles in the lower portion, not in association with the arrector pili muscle. Scanning electron microscopy of the same specimens demonstrated accordion-like structures below the sebaceous gland in the lower half of anagen terminal hair follicles and surrounding the entire circumference of the follicles. Sawtooth-like structures were unilaterally observed at the level of the middle or lower portion below the sebaceous gland. With transmission electron microscopy, these structures were seen as undulations of basal cells which preserved hemidesmosomes, although the basal lamina had been peeled off during the extraction procedure. Thus, this accordion-like structure is a genuine structural variation of some human anagen hair follicles and not an artifact.

Basement Membrane↗
PubMed2000

Luminal localization of blood-brain barrier sodium, potassium adenosine triphosphatase is dependent on fixation.

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

Animals↗
PubMed2004

A molecular mechanism of formalin fixation and antigen retrieval.

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.

Amino Acid Sequence↗
PubMed1996

Acid phosphatase activity in the chick's inner ear.

The cellular localization of distribution of acid phosphatase (AP) in the plastic sections of newly hatched chick's inner ear were investigated utilizing an azo-coupling method. AP activity as evidenced by azo dye deposits were well defined and localized in various cells of membranous labyrinth. Intense AP activity was detectable in the supranuclear area of hair cells in the basilar papilla and vestibular sensory hair cells. As in the case of the other sites of AP activity, marked AP activity was seen in the supranuclear area of the transitional epithelia of crista ampullaris and in the supranuclear area, or diffusely in the cytoplasm of the dark cells at the base of crista ampullaris. The columnar cells and the cells of tegmentum vasculosum showed moderate to strong AP activity. The statoacoustic and vestibular ganglion cells showed various degrees of AP activity. On the AP activity of statoacoustic or vestibular ganglion, in comparison between the sections from JB-4 Plus embedded specimens and those from LR White embedded specimen, the latter could more intensely demonstrate AP activity than the former. Moreover, sections fixed in 2.5% paraformaldehyde demonstrated more intense AP activity than those fixed in 2.5% glutaraldehyde.

Acid Phosphatase↗
PubMed1993

Histological techniques to improve testing of pulpal response of teeth to filling material.

Two fixation fluids, two fixation techniques and two embedding methods were investigated for their effects on the quality of sections of teeth for pulpal response to filling materials to improve evaluation of pulpal responses. Sections from 32 baboon teeth were prepared, half with experimental cavities and half without, using either 10% formaldehyde or 4% glutaraldehyde, longitudinal tooth splitting or removal of the tooth apex, and paraffin or K plast resin embedding; decalcification in a formic acid mixture was a constant throughout. Histometric analysis showed that paraffin embedding produced less shrinkage than the K Plast resin embedding although the difference was not statistically significant. Six parameters of separation at the pulp:dentine interface were studied: embedding, fixative, presence or absence of a cavity, cutting technique and individual animal tooth type. Statistical investigation revealed that fixative, cutting technique, and fixative and cutting technique combined had significant influences on the separation artifact. Of the combinations tested the choice of embedding method depends on which of the two artifacts, shrinkage or separation, is more adverse in the opinion of the investigator. Four percent glutaraldehyde together with the longitudinal split technique of fixation, processed by either K Plast resin embedding or paraffin embedding produced satisfactory pulpal sections.

Animals↗
PubMed1996

Tannic acid supplemented fixation improves ultrastructural evaluation of respiratory epithelium in children with recurrent respiratory tract infections.

Samples of the respiratory mucosa of children with recurrent respiratory infections suspected of having primary ciliary dyskinesia are routinely fixed with glutaraldehyde before ultrastructural examination. This standard technique, however, may not be optimal for visualizing ciliary components or for preserving several cellular and extracellular structures during dehydration and embedding procedures. In this study, brushes of nasal (28 samples) and/or tracheal (9 samples) mucosa from 32 children with recurrent respiratory tract infections were examined. Twenty-nine samples were fixed with glutaraldehyde supplemented with tannic acid to determine if the ultrastructural analysis of respiratory epithelium and bronchial secretions could be improved. Eight samples were conventionally fixed with glutaraldehyde alone. Lesions of the cellular membrane and damaged cells were easily visualized using tannic acid supplemented fixation. Internal ciliary structures including individual microtubules and dynein arms were also more clearly observed. In addition, the internal structure of microvilli of the respiratory epithelium could be studied and the presence of phospholipid-rich surfactant-like material within nasal and tracheal secretions were visualized after tannic acid supplemented fixation. We suggest that addition of tannic acid during fixation is useful for accurate ultrastructural evaluation of respiratory mucosa in both clinical and experimental situations.

Adolescent↗
PubMed2000

Variant chromophobe renal cell carcinoma.

Separation of renal cell tumors into different prognostic groups is an imperative function of the diagnostic pathologist. Recently, chromophobe renal carcinoma has been described as a tumor that is morphologically distinct from conventional "clear cell" carcinoma and that has a low metastatic potential. Identification is based on routine light microscopic features and is confirmed by special stains, immunohistochemistry, and electron microscopy. We present a variant of chromophobe renal carcinoma that did not show the typical cytomorphologic features on light microscopy after formaldehyde fixation. After fixation in Solufix (a commercial fixative), these features were recognized and the diagnosis was confirmed. The tumor also showed an unusual form of calcification and psammoma body formation not previously recognized in chromophobe tumors. Molecular biological assessment was inconclusive, but excluded a chromosome 3p deletion usually found in conventional renal carcinoma. The use of a different primary fixative may provide a cost-effective screening tool to detect variant renal tumors and may have important prognostic implications.

Aged↗
PubMed1976

Pharmacologic aspects of pentamidine.

Pentamidine is an aromatic diamidino compound synthesized originally for the therapy of trypanosomiasis. The pharmacologic effects of pentamidine vary, depending on its route of administration. In animals, the dominant effects have been a precipitous, transitory drop in blood pressure after injection and renal toxicity following repeated administration. To avoid the possibility of immediate toxic reactions associated with iv administration, we now usually give the drug im to humans. Further interest in pentamidine has been stimulated by its usefulness in the treatment of interstitial pneumonia caused by Pneumocystis carinii. In some patients receiving antineoplastic or immunosuppressive therapy who have superimposed P. carinii pneumonia, pentamidine may cause serious renal toxicity. Distribution and excretion studies in animals indicate pentamidine is deposited in tissues, with the greatest concentration in the kidneys, and gradually eliminated over a prolonged period. The mechanism of action of pentamidine against P. carinii or the means whereby fixation in tissues and subsequent toxicity occur have not been elucidated. Recent investigations to help clarify these points indicate that pentamidine inhibits dihydrofolate reductase in all tissues studied both in vitro and in vivo. In addition, pentamidine interacts and forms water-insoluble products with specific nucleotides and nucleic acids.

Amidines↗
PubMed1994

Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study.

A fixative composed of 1% w/v osmium tetroxide dissolved in a tluorocarbon (FC-72) was used to perfusion-fix rat blood vessels for this ultrastructural study. With non-aqueous fixation, the lumenal surfaces of endothelial cells in cardiac and skeletal muscles retained an extracellular coat which was morphologically consistent with glycocalyx. Venules displayed the greatest density and depth (up to 870 nm thick) of glycocalyx, but all vessel types had at least 70 nm of surface coat. The results indicate that non-aqueous fixatives will be useful in studies of the endothelial cell lumenal surface, while offering excellent preservation of general ultrastructure.

Animals↗
PubMed1996

Use of CD23 (BU38) on paraffin sections in the diagnosis of small lymphocytic lymphoma and mantle cell lymphoma.

The CD23 antigen is a low-affinity immunoglubulin E receptor that is expressed during B-cell activation. Recently, it has been shown to be of diagnostic utility in distinguishing between small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL), two entities that can have similar morphologic and immunophenotypic features. Such studies, however, generally required viable cells in cell suspension or cryostat sections for detection of CD23. We evaluated staining for the CD23 antigen in paraffin sections, using BU38, an antibody that detects a fixation-resistant epitope of the antigen. We analyzed 44 SLLs, 3 lymphoplasmacytoid lymphomas, and 39 MCLs. Staining was performed on formalin- or B5-fixed paraffin-embedded tissue sections using L26 (CD20), CD3, Leu22 (CD43), and BU38 (CD23) antibodies. All of the cases were of B-cell phenotype (CD20+), and 42/44 SLLs, 3/3 lymphoplasmacytoid lymphomas, and 33/39 MCLs coexpressed the CD43 antigen. CD23 was positive in 41 (93%) of 44 SLLs. The majority of neoplastic cells (75% or more) stained positively, with a membranous pattern of staining. The staining was moderate in intensity and easily interpreted. Only 1/39 MCLs and 1/3 lymphoplasmacytoid lymphomas were CD23 positive. CD23-positive follicular dendritic cells were, however, present in all of the MCLs, either in residual follicles or in large, disordered meshworks. These results demonstrate that the BU38 antibody can detect CD23 on the cells of SLLs in paraffin sections and that this antibody can have diagnostic utility in routine diagnosis.

Biomarkers, Tumor↗