The Histochemical Journal最新文献

Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semi-quantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p < or = 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p < or = 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.

Accurate ultrastructural localization of the components of centrosomes is an important step toward the determination of their function. We have used an electron microscopy procedure to preserve centrosome-associated antigens which enables their high-resolution localization. The unique part of our procedure is the application of a post-sectioning fixation step which overcomes the poor section contrast and morphological appearance that limits the use of low-temperature processing and Lowicryl embedding. The efficacy of our approach is demonstrated by the efficient labelling of alpha-tubulin in the well-preserved and contrasted microtubule barrels of the centrides of isolated mammalian centrosomes.

The monoclonal antibody MRG-1 has been evaluated for the immunohistochemical detection of the type 3 chain of blood group A in human normal bladder epithelium and bladder tumours. Light microscope examination of paraffin sections demonstrated that this antigen was present in normal epithelium and superficial bladder tumour in patients with blood group A or AB, but was absent in the invasive type of bladder tumour. In normal epithelium, the plasma membrane was positive for this antigen, and the cytoplasm was diffusely stained. In superficial transitional cell carcinoma, the plasma membrane was negative, whereas the cytoplasm was intensely stained in the perinuclear region. This pattern was different from that observed for type 1 and 2 group A antigen, which was recognized mainly at the plasma membrane. However, in superficial transitional cell carcinoma, the staining was also seen on the plasma membrane. The pattern of the localization of this antigen in this carcinoma was influenced by the treatment of organic solvents. Electron microscopical observations confirmed that this antigen was localized on the plasma membrane and also in the Golgi apparatus of the superficial tumour. These results proved that the type 3 chain of blood group A is present in human bladder epithelium and low grade tumours in correspondence with the blood type, but disappears in tumours with high malignant potential. However, its expression is independent of the expressions of the other subtypes which have been studied. Furthermore, the changes in the staining pattern caused by pretreatment with organic solvents suggested possible differences in the microenvironment of the glycolipids containing this type of sugar chain.

The distribution and relative catalytic activities of five plasma membrane enzymes (alkaline phosphatase, dipeptidyl peptidase IV, gamma-glutamyl transpeptidase, microsomal alanyl aminopeptidase and glutamyl aminopeptidase) were examined in human and pig oesophagus. In both species, alkaline phosphatase activity occurred in basal and suprabasal cells of the epithelium and in capillaries. Stromal cells in the human submucosa were particularly reactive. Dipeptidyl peptidase IV was present in blood vessels and capillaries in man and pig and in submucous glands in the pig. The enzyme was also present in both species in the lamina propria cells immediately adjacent to the epithelial basal lamina. In the human, gamma-glutamyl transpeptidase occurred in the epithelial basal cells and in isolated basal and lower prickle cells in the pig. Stromal cells in the human submucosa were strongly reactive and capillaries in the muscularis propria in both species moderately active. Microsomal alanyl aminopeptidase was detected in lamina propria cells adjacent to the epithelial basal cell layer in man and pig and at the apices of mucous cells in pig submucous glands. Weak glutamyl aminopeptidase activity was confined to capillaries in both species. The findings of this study, along with the ready availability of pig oesophagus, suggest that the pig may be a suitable model for studies of the gullet in man.

The germinal centre forms a specialized microenvironment thought to play a key role in the induction of antibody synthesis, affinity maturation of B cells and memory B cell formation. Clonal-expanded follicular B lymphocytes with mutated antigen receptors (centrocytes) have to be selected on the basis of their capacity to compete for binding to antigen held in limited amounts on the follicular dendritic cells. In this way, only high-affinity B cells are selected. Binding to a follicular dendritic cell is an unconditional prerequisite for centrocytes to survive. Cells that do not succeed in binding to a follicular dendritic cell die rapidly by apoptosis. Apoptosis is a common form of cell death characterized by the activation of an endonuclease culminating in nuclear destruction. The pathway by which apoptosis is triggered varies from cell type to cell type. However, for germinal centre B cells this process is still poorly understood.

Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.

A simple procedure is described for removing Epon resin from semi-thin 1 micron sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4-7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.

Recently, we observed that atrial natriuretic peptide (ANP) immunoreactivity was present in Purkinje fibres and nerve fibre varicosities in the conduction system of the bovine heart. In order to elucidate further the morphological correlation between natriuretic peptides and the conduction system, the distribution of brain natriuretic peptide (BNP) was examined. The different parts of the conduction system in the bovine heart were dissected out and processed for immunohistochemistry with antisera against BNP and ANP. BNP immunoreactivity was frequently observed in Purkinje fibres of the atrioventricular bundle, whereas only a few Purkinje fibres in the ventricular part of the conduction system showed immunoreaction. BNP immunoreactivity was detected in regions of the Purkinje fibres which also showed ANP immunoreactivity. BNP immunoreactivity was not observed in nerve fibre varicosities. Methodologically, a larger number of small BNP immunofluorescent granular structures was observed by using an elution-restaining technique instead of conventional immunohistochemistry. The present study shows that BNP and ANP immunoreactivities frequently occur in the atrioventricular bundle and that they are co-localized in Purkinje fibres, but not in nerve fibre varicosities, in the conduction system. As previously has been proposed for ANP, the present observations suggest that also BNP may act in an autocrine and/or paracrine way in the conduction cells.

The pattern of glycan distribution in the early human yolk sac has been investigated using a panel of lectins. Two 6-week and one 8-week human yolk sacs, and one 8-week fetal liver from live, ectopic pregnancies were fixed and embedded in epoxy resin. Lectin histochemistry was carried out on sections of these tissues using 23 biotinylated lectins and an avidin-biotin peroxidase revealing system. Mesothelial surfaces expressed most subsets of N-glycans (other than high mannose types), N-acetyl-lactosamine, sialic acid, and alpha 1,6-N-acetylgalactosamine. Endodermal surface and lateral membranes resembled those of mesothelium, but showed a preponderance of alpha 2,6-sialyl residues. Most intracellular granules contained N-glycan. There was a marked heterogeneity of granules in the endodermal cells, with different subsets varying in both staining and positional characteristics. The mesenchymal matrix bound most of the lectins used in the study, and expressed fucosyl residues which were also detected in the endothelium. Fetal liver parenchyma showed very similar staining patterns to those seen in the endoderm except for the distribution of N-acetylglucosamine, which was sparse. Despite some common features, each germ cell layer had a distinct 'glycotype', with some saccharides showing extreme topographical restriction.

In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.