Histochemistry最新文献

The M. urethralis was morphologically investigated in ten medium-sized female dogs of different breeds and age, as well as histochemically and stereologically analysed in a homogeneous group of five female beagles. Macroscopically, the muscle was essentially confined to the distal third and most strongly developed in the fourth quarter of the urethra. Here, it surrounded the urethra transversely at the ventral and lateral aspects, passing with its caudal fibres dorsally onto the vagina. The muscle fibres were assembled in groups of different sizes and usually separated by thick connective tissue septae. Based on the myofibrillar actomyosin ATPase (mATPase) reaction, type I and two main subtype II fibres could be differentiated. Type II fibres were, however, indistinguishable by their metabolic enzyme activities since both subclasses displayed oxidative-glycolytic properties. The subtype II fibres containing the more acid-labile mATPase activity were classified as IIA, whereas the other main subtype was designated IIS (subtype) and considered as peculiar to the dog. In addition, the investigation revealed a rare fibre type exhibiting the histochemical profile of IIC fibres. There was no evidence of classical glycolytic IIB fibres. The M. urethralis was composed of 24% type I and 76% type II fibres with an average diameter of 34.9 and 28.5 microns, respectively. Connective tissue constituted 52.8% of the total muscle volume. Due to the predominance of fast twitch II fibres, the urethral muscle is not designed to maintain a high tone over long periods of time. Its primary role is to function rapidly and intermittently guaranteeing urinary continence during stress situations when other continence factors are overburdened.

Detection of DNA synthesizing cells may often be achieved by immunocytochemical detection of bromodeoxyuridine (BrdU), which is rapid and appears to give similar results to those found using tritiated thymidine. However, the methodology for detection of BrdU involves a denaturation or digestion step to allow access of the antibody to BrdU incorporated into single- rather than double-stranded DNA. We wished to determine if microwave treatment could be used to enhance the detection of BrdU without the need for any other digestion/denaturation steps. An important consideration was to investigate whether such treatment produces a similar quantitative result, since BrdU detection is usually assessed on the basis of cell number rather than topographical distribution. We have found that microwave treatment can allow considerably lower antibody concentrations and eliminates the need for any other denaturation step. It also reduces the non-specific background staining found when using monoclonal antibodies on mouse tissue. We have performed cell counts and found that the number of BrdU positive cells remains constant for a range of different immunocytochemical parameters. We also report conditions where immunopositivity is adversely affected by changes in technique and describe the optimised conditions for obtaining reproducible results.

Alveolar type 2 cells are known to take up surfactant phospholipids and proteins from the alveolar space and recycle them into secretory organelles via a receptor-mediated endocytic pathway. To clarify the intracellular route(s) through which materials ingested by the cells are processed, we examined the immunocytochemical localization of late endosomal and lysosomal membrane markers, rab 7 and lamp 1 proteins, within rat alveolar type 2 cells. The limiting membranes of lamellar bodies (LBs) showed positive immunoreactivity for both proteins, whereas multivesicular bodies (MVBs) exhibited positive immunoreactivity only for lamp 1 protein on free vesicles in the MVB lumen. From these findings, it is suggested that LBs are not only secretory granules, but also constitute one of the late endosomal compartments of the cells and that MVBs of this cell type may be targeted to cell organelle(s) other than lysosomes.

The proliferative activities of the different cellular compartments of the developing mouse ovary, uterus, and oviduct were studied by radioautographic assessment of DNA synthesis with [3H]-thymidine labeling and by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). The distributions of estrogen and progesterone receptors (ER and PR) were studied by immunohistochemical staining. The values of the PCNA positive staining indices were a little higher than that of the radioautographic labeling indices. However, linear relations were shown for the two indices. The proliferative activities were high from postnatal day 1-7 and decreased from day 14 in the different cellular compartments of the ovary. The proliferative activities were high on days 1, 3 and decreased from day 7 in the uterus and oviduct. Staining of ER and PR was very weak in the surface epithelium, stroma and large follicles of the ovary. Positive staining for ER occurred from day 14 in the uterine epithelium and from day 7 in oviductal epithelium. Positive staining for PR was observed from day 1 in both the uterine and oviductal epithelium. However, the positivity of both ER and PR occurred from postnatal day 1 in the stromal cells of the uterus and oviduct. These results suggest that the appearance of the steroid receptors differ between the different cellular compartment of the reproductive organs. The proliferative activities have an inverse relation with the expression of the steroid hormone receptors in the female reproductive organs during developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Two monoclonal antibodies of types IgG2b and IgG2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N1-acetylspermidine (N1-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.

The role of oxidative stress resulting from production of reactive oxygen species and/or from suppression of the cellular antioxidant capacity in parasitic infections is shortly reviewed. The experimental part of the paper deals with the glutathione (GSH)--glutathione reductase (GR) system, a cornerstone of intracellular antioxidant defence mechanisms. For studying this system in parasitic diseases such as malaria new or modified methods are required. Total glutathione comprising GSH and glutathione disulphide (GSSG) in blood samples was assayed as follows. One volume of blood (> or = 10 microliters) is mixed with two volumes of 5% sulphosalicylic acid; after centrifugation (5 min, 10000 g), 10 microliters of supernatant is taken for spectrophotometric analysis using the 5,5'-dithiobis(2-nitrobenzoate) (DTNB)-glutathione recycling assay. When compared with the original method, the procedure reported here is more sensitive, less time-consuming, avoids unfavourable pH-values and leads to a sample which when frozen is stable for months. In a pilot study, the method was applied to 14 patients suffering from malaria caused by Plasmodium falciparum. The concentrations of erythrocyte glutathione were significantly decreased in the patients (1.42 +/- 0.47 mM, mean +/- SD) when compared to age- and sex-matched controls (2.11 +/- 0.45 mM, P < 0.01). The findings are contrasted with P. falciparum cultures in vitro where glutathione levels are known to be elevated. Based on the characteristics of GR a concept of determining the redox state of single cells is introduced.(ABSTRACT TRUNCATED AT 250 WORDS)

A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV with scanned areas of 200 x 400 microns. The semi-quantitative data indicated that in LPS-treated groups both adhesion molecules were expressed at a significantly higher level than in all other groups (P < 0.01). In addition, after a 4 h treatment the expression levels of E-selectin in all groups were higher compared to ICAM-1. The experimental data from X-ray microanalysis were compared with data obtained from an enzyme-linked immunosorbent assay (ELISA) and similar values were found for both types of preparation. This microanalytical method is relatively simple and seems to be suitable for immunogold labelling studies on different types of endothelial cells in vitro.

Evidence for the presence of peptides, related to insulin-like growth factor 2 (IGF-2), has been obtained in the endocrine pancreas of the elasmobranchian species Raja clavata, the sting ray. By radioimmunoassay, IGF-2-like immunoreactivity was detected in Raja pancreas extract. Further characterization of this activity by acid gel chromatography revealed two distinct peaks of IGF-2-like immunoreactivity with apparent molecular weights of approximately 8.2 kDa and 4.5 kDa. Using the same IGF-2 antibody as well as antisera specific for mammalian IGF-1, insulin, glucagon, somatostatin and pancreatic polypeptide in double immunofluorescence studies, IGF-2-like immunoreactivity was located exclusively in insulin-immunoreactive cells. In contrast, IGF-1-like immunoreactivity was mainly observed in somatostatin- and glucagon-immunoreactive cells. A varying proportion (0-70%) of insulin-immunoreactive cells, however, displayed both IGF-1- and IGF-2-like immunoreactivity. Absorption studies indicated that the IGF-2-like peptides in Raja are different from mammalian and submammalian insulin and mammalian IGF-1, but similar to mammalian IGF-2. Thus, IGF-2-like peptides seem to occur during evolution as early as the phylogenetic development of the elasmobranchians. Furthermore, the results indicate a particularly conservative evolution of the islet IGF-2 system.

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NA-DPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.

The colocalization of surfactant protein A (SP-A) and the alveolar macrophage markers ED1 and RM-1, as well as various lectins of the N-acetyl-galactosamine group [Maclura pomifera lectin (MPA), Dolichos biflorus lectin (DBA), soybean agglutinin (SBA)] and of the mannose group [Canavalia ensiformis lectin (ConA), Galanthus nivalis lectin (GNA)] was studied in normal and fibrotic rat lung tissues. In normal tissue, SP-A was located preferentially in the alveolar macrophage subpopulation lacking specific binding sites for lectins of the N-acetylgalactosamine group (DBA and SBA), although 50% of MPA-binding macrophages contained SP-A. The ED1-positive cells were SP-A-negative, whereas SP-A uptake could be detected among the RM-1 immunoreactive as well as the ConA and GNA binding macrophages. In fibrotic lung tissue, however, a small number of DBA and SBA binding macrophages contained SP-A and the percentage of GNA and ConA binding alveolar macrophages exhibiting SP-A immunoreactivity was reduced. Additionally, the number of ED1+/SP-A+ macrophages was found to be increased. Immunoelectron microscopy revealed accumulation of SP-A in the extracellular space. The differing SP-A content in different alveolar macrophage subpopulations suggests a more complex mechanism of uptake and degradation of surfactant proteins in normal and pathological conditions, which cannot simply be explained by the glycoconjugate pattern on the surface of alveolar macrophages.