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PubMed1988

Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semi-thin sections.

We have designed a procedure to investigate vasopressin (AVP) gene expression on plastic-embedded tissue by using in situ hybridization to detect AVP mRNA and immunohistochemistry to detect AVP. Rat brain was fixed and vibratome slices were incubated with a 45-base synthetic oligonucleotide complementary to AVP mRNA labeled with 35S, embedded in Araldite, and cut into semi-thin serial sections that were either processed for autoradiography or treated with an AVP antiserum. The results show that AVP mRNA is detectable in magnocellular neurons of the supraoptic and paraventricular nuclei in both vibratome and semi-thin sections. Osmication after hybridization does not modify the signal. AVP mRNA is restricted to the cytoplasm of magnocellular neurons and to the proximal portion of certain processes. Neurons labeled with the AVP probe were also stained with the AVP antiserum. AVP mRNA quantity and the intensity of AVP immunoreactivity are not consistently related in neurons. At least two hypotheses must be considered to explain these differences: first, the procedure presently used could lead to a reaction intensity that does not exactly reflect the amount of antigen or mRNA present in cells; second, the difference observed may reflect the fact that transcriptional and translational events are not constantly linked and can be regulated differently from one AVP neuron to another. This method provides a way to detect mRNA on semi-thin sections together with antigenic molecules and to accurately investigate gene expression in complex tissues with optimal histological quality.

Animals↗
PubMed1990

BrdUrd labeling of S-phase cells in testes and small intestine of mice, using microwave irradiation for immunogold-silver staining: an immunocytochemical study.

In this study, BrdUrd labeling of S-phase cells in the small intestine and testes was accomplished using microwave irradiation. In this way crypt cells, spermatogonia, and Leydig cells could be labeled using removable plastic-embedded sections and immunogold-silver staining (IGSS). By using short periods of microwave irradiation for incubation of the monoclonal antibodies and the protein A-colloidal gold solution, the detection of BrdUrd-labeled cells could be remarkably enhanced. A comparative study of BrdUrd labeled spermatogonia in the testis of a Cpb-N mouse that received both [3H]-thymidine and BrdUrd proved that 90% of the BrdUrd-labeled cells also showed [3H]-thymidine labeling. The radioactive [3H]-thymidine labeling was a time-consuming method of 4 weeks' duration, whereas the BrdUrd-labeled cells could be labeled, fixed, enhanced, and counterstained in less than 3 hr. This investigation proves that BrdUrd labeling of S-phase cells can be a reliable, reproductive, rapid, and non-radioactive alternative method for [3H]-thymidine labeling of proliferating cells.

Animals↗
PubMed1990

Improved immunocytochemical staining of carcinogen-DNA adducts by a capillary slot block system.

We developed an immunocytochemical protocol in which incubation occurs in a capillary slot instead of the conventional horizontal drop. Slots of constant width were formed by placing slides on top of each other with parafilm spacer layers in between. Cryostat or semi-thin plastic-embedded sections were cut from organs of carcinogen-treated experimental animals. Carcinogen-DNA adducts were visualized in the affected nuclei by a double peroxidase-antiperoxidase method using rabbit antisera specific for certain DNA adducts formed. The staining in capillary slot blocks offered better staining reproducibility than the conventional method. This is particularly important when the staining intensity must be quantified. In addition, handling of the blocks was substantially less laborious than the individual treatment of slides, making this protocol especially suitable for larger series of slides. Other applications for the capillary slot block protocol should be enzyme histochemistry and in situ hybridization.

Animals↗
PubMed1992

Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues.

A new method is described for locating DNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing terminal deoxynucleotidyl transferase (TdT) and various non-isotopic nucleotide analogues. The labeled nucleotides bound to the surface of ultra-thin sections were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern was strongly dependent on the divalent cation used in the TdT medium. The method revealed with great precision the specific DNA-containing structures within Ehrlich tumor cells, even where DNA was present in very low amounts. The method is compatible with all usual fixation and embedding procedures and can be combined with cytochemical methods. The in situ TdT method provides a very useful tool for pinpointing the precise location of DNA within biological material at the ultrastructural level.

Animals↗
PubMed1992

Effects of tannic acid on antigenicity and membrane contrast in ultrastructural immunocytochemistry.

We have modified our previous method for immunogold staining of unosmicated, plastic-embedded tissue by addition of tannic acid as a post-fixative to increase membrane contrast. Overall cell ultrastructure and organelle membranes, in particular, appeared well preserved after this treatment. We evaluated quantitatively the effect of tannic acid on the antigenicity of several membrane proteins in rat liver and intestine. For all antigens tested, significant antigenicity was retained on both intracellular and plasma membranes. However, the level of antigenicity decreased with increased concentrations of tannic acid. This effect was most apparent on the apical and basolateral membranes of hepatocytes and on the apical membrane of enterocytes, surfaces that had been in direct contact with the tannic acid fixative. The results indicate that when low concentrations of tannic acid are employed, this method yields greatly enhanced membrane contrast while preserving sufficient antigenicity to facilitate the ultrastructural localization of many membrane and other antigens.

Animals↗
PubMed1993

Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.

Animals↗
PubMed1994

Novel in situ double labeling for simultaneous detection of proliferation and apoptosis.

We describe a novel double-labeling method to simultaneously investigate proliferation and apoptosis from plastic-embedded biopsy specimens (PEBs). Infusions of bromo- and/or iododeoxyuridine (BrdU/IudR) were given to 10 patients, five with acute myeloid leukemia (AML) and five with myelodysplastic syndromes (MDS), and S-phase cells were measured in PEBs using a monoclonal anti-IudR/BrdU antibody. Apoptosis was measured by in situ end-labeling (ISEL) of DNA. The results demonstrate that both AML and MDS are highly proliferative disorders but that there is almost no apoptosis in the former, whereas extensive apoptosis was observed in the latter. Double labeling revealed that large numbers of S-phase cells in MDS were simultaneously undergoing apoptosis. We conclude that the high cell death in MDS cancels the high cell birth, resulting in a functionally aplastic marrow and thus accounting for the observed ineffective hematopoiesis. On the other hand, AML is rapidly fatal, probably owing to high cell birth with no or minimal cell death. Therapeutic strategies to prevent intramedullary programmed cell death of hematopoietic precursors should be evaluated in MDS, and efficacy of chemotherapy in AML can be assessed by measuring the induction of apoptosis in post-treatment biopsy specimens.

Acute Disease↗
PubMed1995

Bromodeoxyuridine immunohistochemistry of epon-embedded undecalcified bone in a canine fracture healing model.

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry of paraffin-, methyl methacrylate (MMA)-, and epon-araldite (epon)-embedded canine bone specimens to establish an optimal technique for studying cell kinetics of fracture healing in a canine tibial gap model. Dogs were sacrificed 4 months after tibial ostectomy and 1 hr after i.v. injection of BrdU (100 mg/kg). BrdU immunohistochemical staining with a peroxidase-labeled streptavidin-biotin (LAB-SA) method was performed on thin sections of tibia fixed in 70% ethanol and embedded in paraffin, MMA, or epon. Thin section of small intestines fixed in 70% ethanol and 10% neutral buffered formalin and embedded in paraffin, MMA, or epon were BrdU-stained and served as a model for proliferating tissue. Good and consistent BrdU immunostaining without detachment of bone sections was obtained for epon-embedded undecalcified bone sections. BrdU-positive cells were easily identifiable, in contrast to negligible background staining. BrdU-labeled osteoprogenitor cells and osteoblasts were observed around and on the surface of woven bone in external and internal callus of the ostectomy gap. Nuclei of osteocytes were not labeled. In contrast to the epon-embedded specimens, BrdU immunostaining of paraffin-embedded decalcified and MMA-embedded undecalcified bone specimens was unsatisfactory. The results of this study suggest that BrdU immunohistochemistry of ethanol-fixed, epon-embedded, undecalcified canine bone sections is a technique suitable for study of fracture healing with the described methodology.

Animals↗
PubMed2005

The effect of cartilage and bone density of mushroom-shaped, photooxidized, osteochondral transplants: an experimental study on graft performance in sheep using transplants originating from different species.

BACKGROUND: Differences in overall performance of osteochondral photooxidized grafts were studied in accordance of their species origin and a new, more rigorous cleansing procedure using alcohol during preparation. METHODS: Photooxidized mushroom-shaped grafts of bovine, ovine, human and equine origin were implanted in the femoral condyles of 32 sheep (condyles: n = 64). No viable chondrocytes were present at the time of implantation. Grafts were evaluated at 6 months using plastic embedded sections of non-decalcified bone and cartilage specimens. Graft incorporation, the formation of cyst-like lesions at the base of the cartilage junction as well as cartilage morphology was studied qualitatively, semi-quantitatively using a score system and quantitatively by performing histomorphometrical measurements of percentage of bone and fibrous tissue of the original defects. For statistical analysis a factorial analysis of variance (ANOVA- test) was applied. RESULTS: Differences of graft performance were found according to species origin and cleansing process during graft preparation. According to the score system cartilage surface integrity was best for equine grafts, as well as dislocation or mechanical stability. The equine grafts showed the highest percentage for bone and lowest for fibrous tissue, resp. cystic lesions. The new, more rigorous cleansing process decreased cartilage persistence and overall graft performance. CONCLUSION: Performance of grafts from equine origin was better compared to bovine, ovine and human grafts. The exact reason for this difference was not proven in the current study, but could be related to differences in density of cartilage and subchondral bone between species.

Animals↗
PubMed1979

Kniest dysplasia. A histochemical study of the growth plate.

Chondro-osseous tissue from four patients with the Kniest dysplasia was studied histochemically using a new plastic embedding technique. Extensive vacuolar changes were observed p--1 throughout the endochondral growth plate and adjacent resting cartilage. These changes occurred within the cartilage matrix and also in the lacunae of degenerating chrondrocytes. The septa of the lesions contained chondroitin sulfate, but little keratan sulfate or collagen. Resting cartilage not adjacent to the growth plate stained irregularly and showed few of the vacuolar lesions, and chondrocytes were enlarged and contained cytoplasic inclusions, but no vacuolar material. Thus, there appears to be a sequence of events initiated by cellular accumulation of a substance and progressing to cellular and matrix degeneration.

Adolescent↗
PubMed1996

Paget's disease is associated with changes in the immunohistochemical distribution of noncollagenous matrix proteins in bone.

Paget's disease of bone is characterized histologically by abnormal architecture of bone matrix. Extensive areas of woven bone and numerous scalloped cement lines occur as a result of increased irregular remodeling. Noncollagenous proteins (NCP) play an important role in the organization and mineralization of bone matrix and promote distinct cell-matrix interactions necessary for normal remodeling. To gain insight into the pathological changes in the biochemical composition of Pagetic bone, the distribution of NCPs in the calcified matrix of bone from patients with known Paget's disease was compared to that of bone from normal healthy volunteers. Undecalcified plastic-embedded sections of bone were stained immunohistochemically using antibodies generated against several NCPs. In Pagetic and normal bone a similar distribution of osteopontin was observed at cement (reversal) lines, whereas significant differences were observed in the distribution of osteopontin in the matrix immediately adjacent to Haversian canals, where initial osteoclast recruitment and attachment occur. The differences in osteopontin distribution appeared to be related to the state and severity of the disease. Site-specific differences in the distribution of osteonectin, osteocalcin, and decorin were also observed between normal bone and cortical and periosteal de novo Pagetic bone, whereas the distribution of other matrix proteins, such as biglycan, was unchanged. We conclude that these site-specific changes in the biochemical distribution of NCPs in Pagetic bone probably reflect abnormal production and/or incorporation during bone remodeling and may lead to disorganized matrix assembly and mineralization as well as have profound effects on bone cell functions.

Adult↗
PubMed1987

Cytoarchitecture of the Xenopus thymus following gamma-irradiation.

This paper describes in vitro and in vivo attempts to deplete the 4- to 8-month-old Xenopus laevis (J strain) thymus of its lymphocyte compartment. Gamma irradiation (2-3000 rad) of the excised thymus, followed by two weeks in organ culture, is effective in removing lymphocytes, but causes drastic reduction in size and loss of normal architecture. In contrast, in vivo whole-body irradiation (3000 rad) and subsequent in situ residence for 8-14 days proves successful in providing a lymphocyte-depleted froglet thymus without loss of cortical and medullary zones. In vivo-irradiated thymuses are about half normal size, lack cortical lymphocytes, but still retain some medullary thymocytes; they show no signs of lymphocyte regeneration when subsequently organ cultured for 2 weeks. Light microscopy of 1 micron, plastic-embedded sections and electron microscopy reveal that a range of thymic stromal cell types are retained and that increased numbers of cysts, mucous and myoid cells are found in the thymus following whole-body irradiation. In vivo-irradiated thymuses are therefore suitable for implantation studies exploring the role of thymic stromal cells in tolerance induction of differentiating T lymphocytes.

Animals↗
PubMed1987

Advances in oto-immunology: new trends in functional pathology of the temporal bone.

By using monoclonal and polyclonal antibodies against cell-membrane receptors and cytoskeletal proteins, the cellular and tissue composition of any organ can be far better determined. This is illustrated in the tympanic membrane, middle ear mucosa, and cholesteatoma. Immunotechnology applied to the temporal bone is shown with an animal-ototoxicity model. Antibodies to keratin and vimentin (i.e., to proteins of the fibrillar components that make up the cytoskeleton) stain specific tissue compartments. Langerhans' cell and T-lymphocyte subsets predominate in a cholesteatoma. The normal human tympanic membrane is devoid of these cells. Gentamicin can be traced with a polyvalent anti-gentamicin antiserum in serial sections of the cochlea and kidney. The drug accumulates in the outer hair cells and proximal tubular cells, respectively. A new technique of CT-scanning, microslicing, plastic embedding, and semithin sectioning of undecalcified human temporal bone is described. Perspectives in applying immunotechnology to this method of human temporal bone processing are discussed.

Animals↗
PubMed1992

Neural regeneration in the noise-damaged chinchilla cochlea.

Recent studies in the bird ear have shown that degenerated hair cells are sometimes replaced by regenerated receptor cells. The present study evaluated the adult mammalian cochlea for evidence of hair-cell and nerve-fiber regeneration. Eighty-eight noise-damaged chinchilla cochleas were examined as plastic-embedded whole mounts by phase-contrast and bright-field microscopy. No signs of hair-cell regeneration were found. However, 32 (70%) of 46 cochleas damaged by high-intensity noise and 20 (48%) of 42 cochleas damaged by moderate-intensity noise contained a variable number of nerve fibers which appeared to be regenerated. These fibers, which were located in severely damaged areas of organ of Corti, differed from residual fibers with respect to their diameters, the degree and pattern of myelination, and by the abnormal paths they followed within the osseous spiral lamina and on the basilar membrane. The number of regenerated fibers varied with type of exposure and length of recovery. The strongest response was found in ears exposed to a high-intensity, low-frequency noise. The results described here indicate that a potential exists for the biological restoration of the mammalian inner ear.

Animals↗
PubMed1997

The thickness of human vertebral cortical bone and its changes in aging and osteoporosis: a histomorphometric analysis of the complete spinal column from thirty-seven autopsy specimens.

The object of this study was to analyze the cortical thickness (Ct.Th) of the ventral and dorsal shell of the vertebral bodies throughout the human spine in aging and in osteoporosis. Therefore, the complete front column of the spine of 26 autopsy cases (aged 17-90, mean 42 years) without diseases affecting the skeleton and of 11 cases (aged 58-92, mean 77 years) with proven osteoporosis were removed. A sagittal segment prepared through the center of all vertebral bodies was undecalcified, embedded in plastic, ground to a 1 mm thick block, and stained using a modification of the von Kossa method. The analysis included the measurement of the mean cortical thickness of both the ventral and dorsal shell, respectively (from the third cervical to the fifth lumbar vertebral body). The qualitative investigation of the structure of the cortical ring completed the analysis. The presented data revealed a biphasic curve for both the ventral and dorsal shell, skeletally intact with high values of the cortical thickness in the cervical spine (285 microm), and a decrease in the thoracic (244 microm) and an increase in the lumbar spine (290 microm). The mean thickness of the ventral shell is in general greater than the thickness of the dorsal shell in both skeletally normal and osteoporotic cases. The cortical thickness of the spine showed no gender-specific differences (p = NS). There was a slight decrease of the cortical thickness with aging; however, this decrease and the correlation of cortical thickness to age was only significant below vertebral body T8 (r = 0.225-0.574; p(r) < 0.05-0.005). Most interestingly, however, osteoporosis presents itself with a highly significant loss of cortical thickness throughout the whole spine. This decrease of cortical thickness was more marked in the dorsal shell (p < 0.05) than in the ventral shell (ventral from C3 to T6 [p < 0.05] below T6 [p = NS]). We therefore conclude that in osteoporosis the loss of spinal bone mass is not only a loss of trabecular structure but also a loss of cortical thickness. Furthermore, these results may explain the development of regions of least resistance within the spine in aging and the clustering of osteoporotic fractures in the lower thoracic and lumbar spine.

Adolescent↗
PubMed1982

Immunocytochemical and ultrastructural identification of mitotic cells in the pituitary gland of ovariectomized rats.

Light microscopic immunocytochemistry was utilized on plastic-embedded, acrolein-fixed pituitary glands from intact rats and rats killed 14 days after ovariectomy to determine which cell types were undergoing cell division. A significant increase in the number of cell divisions in anterior pituitary cells was seen in ovariectomized rats compared to intact controls. Most of the dividing cells in ovariectomized rats were immunocytochemically identified as gonadotrophs, but dividing somatotrophs and non-immunoreactive cells were also found. All of the dividing gonadotrophs stained with both anti-FSH beta and anti-LH beta. These cells were large and ovoid with plentiful vesiculated rough endoplasmic reticulum and a single population of granules with a mean diameter of 200 nm. Many of the dividing cells which were not immunoreactive with any of the antisera employed contained small granules, 100 nm in diameter, but the content of these granules was not determined.

Animals↗
PubMed2002

Cytokine accumulation in osteitis fibrosa of renal osteodystrophy.

Bone marrow fibrosis occurs in association with a number of pathological states. Despite the extensive fibrosis that sometimes characterizes renal osteodystrophy, little is known about the factors that contribute to marrow accumulation of fibrous tissue. Because circulating cytokines are elevated in uremia, possibly in response to elevated parathyroid hormone levels, we have examined bone biopsies from 21 patients with end-stage renal disease and secondary hyperparathyroidism. Bone sections were stained with antibodies to human interleukin-1alpha (IL-1alpha), IL-6, IL-11, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) using an undecalcified plastic embedding method. Intense staining for IL-1alpha, IL-6, TNF-alpha and TGF-beta was evident within the fibrotic tissue of the bone marrow while minimal IL-11 was detected. The extent of cytokine deposition corresponded to the severity of fibrosis, suggesting their possible involvement in the local regulation of the fibrotic response. Because immunoreactive TGF-beta and IL-6 were also detected in osteoblasts and osteocytes, we conclude that selective cytokine accumulation may have a role in modulating bone and marrow cell function in parathyroid-mediated uremic bone disease.

Adult↗
PubMedDate not supplied

[Mast cell hyperplasia in bone oxalosis].

BACKGROUND: To assess by means of histomorphometry the incidence of bone marrow mast cell hyperplasia in patients with chronic renal failure and oxalosis. MATERIAL AND METHODS: Eighteen individuals were assigned to three groups: 6 (4 males and 2 females, aged 26.31 +/- 2.5 yrs) had chronic renal failure (CRF) and oxalosis of bone; 6 (1 male and 5 females aged 22.1 +/- 3.56 yrs) had CRF and 6 normal (5 males and 1 female aged 23 +/- 2.78 yrs) individuals entered the control group. Quantitative histologic assessments were completed in undecalcified sections of plastic embedded iliac crest bone biopsies stained by the Toluidine Blue method for identification of mast cells. The number of mast cells (cell/mm2 tissue area, x +/- sd) was determined by a semiautomatic image-analyzing system. RESULTS: The number of mast cells was greater in patients with oxalosis of bone, 32.67 +/- 9.59, than in patients with CRF (20.84 +/- 5.04, p < 0.05) and than in the control group (3.26 +/- 1.03, p < 0.001). CONCLUSIONS: Oxalosis of bone is associated with substantial increases in the number of mast cells in the bone marrow. Such a change is not related to chronic renal failure per se and does not appear to represent a non-specific response to bone marrow fibrosis. Mast cell accumulation may contribute to the development of bone marrow fibrosis seen in this disorder.

Adult↗