Histochemistry最新文献

Basolateral uptake of chloride by the HCl-secreting parietal cells of the gastric (oxyntic) glands is most likely mediated by a HCO3-/Cl- anion exchange mechanism. Circumstantial evidence indicates that in rodents the anion exchange proceeds through an anion exchanger 2(AE2)-like membrane protein. In the present study, we raised antibodies against a bacterial fusion protein expressing a approximately 26-kDa portion of the human AE2 sequence. These antibodies were used to identify and localize AE2 in the human stomach. Here we report that the mucosa of the human stomach expresses an approximately 160-kDa immunoreactive form of AE2 containing the AE2-specific exoplasmic domain (Z-loop) as identified by polymerase chain reaction. Immunostaining specific for AE2 was restricted to the basolateral membrane domain of parietal cells and was also detected in small epithelial cells localized in the glandular isthmus region. The latter cells most likely represent pre-parietal cells. Parietal cells were identified by simultaneous and sequential labelling with antibodies against the gastric H+,K(+)-ATPase and actin, respectively. Both actin and the H+,K(+)-ATPase were localized along the apical membrane of parietal cells and the membrane of their secretory intracellular canaliculi. In addition, actin was shown to be colocalized with AE2 along the basolateral cell surface. Discontinuous staining for AE2 coincided with infoldings of the basolateral plasma membrane labelled by the actin antibody. These observations indicate that AE2 might be placed at specialized (folded) microdomains of the basolateral cell surface by linkage to the actin-based cytoskeleton.

Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.

The present study deals with the immunohistochemical localization of S-100 protein in the testes of seven mammalian species including rat, cat, dog, pig, sheep, cattle and horse. Significant differences are demonstrated in the cellular distribution and intensity of immunoreaction for the protein. In bull, ram, boar and cat testes S-100 protein was localized in the cytoplasm and nuclei of Sertoli cells. A particularly intense staining was seen in the modified Sertoli cells of the terminal tubular segment. With the exception of the cat and horse S-100 protein immunoreactivity was additionally found in epithelial cells of the straight testicular tubules and in the epithelial cells of the rete testis. Endothelial cells of capillaries, veins and lymphatic vessels are regularly S-100 immunoreactive in ruminants. Leydig cells were found to be strongly positive for S-100 protein in the cat and rat testes and to a lower degree in pig and horse testes. Finally a distinct immunostaining of peritubular cells was restricted to the testis of dogs and rats. The remarkable species-specific variations of immunoreactivity for S-100 protein in different cell types of the testis support the hypothesis that S-100 protein is multifunctional protein and may have a different function in testicular physiology.

Target cells for 3H-labeled 1 alpha, 25(OH)2 vitamin D3 [1,25(OH)2D3, vitamin D] and its analog 3H-labeled 22-oxa-1 alpha, 25(OH)2 vitamin D3 (OCT) have been identified during endochondral and intramembranous ossification in developing, undecalcified, unembedded bone, using thaw-mount autoradiography. Two-day-old neonatal rats were injected with [3H]1,25(OH)2D3 or [3H]OCT; after 2 h leg, spine, and head were frozen and sectioned. In the epiphyseal-metaphyseal region specific nuclear concentrations of [3H]1,25(OH)2D3 and [3H]OCT were observed in identical cell populations, being low in cells of the articular and resting zone, intermediate in the proliferating zone, and highest in hypertrophic chondrocytes and in osteoblasts and precursor cells. In the primary spongiosa intertrabecular spaces there were a large number of cells with nuclear labeling--probably osteoblasts and precursor cells. In contrast, in the secondary spongiosa intertrabecular spaces, apparent blood-forming cells were mostly unlabeled. Osteoblasts along bone spicules and compact bone in long bones, vertebrae, and head also showed strong nuclear labeling, as did cells of the periosteum. These data suggest that 1,25(OH)2D3 and OCT regulate development, differentiation, and activities of chondrocytes and osteoblasts, including differentiation of resting chondrocytes into proliferating and hypertrophic chondrocytes that involve "chondroclastic" enlargement of lacunae and "trans-differentiation" of surviving hypertrophic chondrocytes; differentiation of stroma cells into osteoblasts; and in periosteum and other regions of intramembranous ossification differentiation of precursor cells and osteoblasts. Nuclear receptor binding and their selective and hierarchical distribution during cell differentiation appear to correspond to multiple genomic effects toward growth, regeneration and repair. The findings indicate a physiological significance and therapeutic potential of 1,25(OH)2D3 and in particular of its less hypercalcemic analog OCT.

Formaldehyde fixation of biopsy specimens for routine purposes has often been held responsible for the poor reproducibility of immunohistochemical studies. Recently, antigen retrieval (AGR) using microwave irradiation was described as a potential tool to enhance immunostaining. A comparison of conventional staining and staining after microwave heating was performed for 52 markers, using tissues fixed in formaldehyde for 24 h, 1 to 6 weeks and 3 years respectively, as well as consultant case material. After adequate duration of fixation (24 h), only a few markers (17%) showed better results after AGR, but this percentage was increased to 50% when tissues were fixed for longer periods. Maximal enhancement was obtained in the group of consultant cases (58% of tested markers demonstrated better staining results), in which the period of fixation and tissue processing was unknown. To achieve reliable enhancement with AGR, continuous heating (100 degrees C) should not be shorter than 20 min. In conclusion, AGR may become the most important tool to simplify and equalize immunohistochemical techniques, if critically evaluated.

Analysis of gene expression using gonadotropin-releasing hormone (GnRH) antisense oligonucleotide confirmed by immunocytochemical localization the occurrence of GnRH neurons along the nervus terminalis in the steelhead trout (Oncorhynchus mykiss). Double-label immunocytochemistry revealed the distribution of mammalian (m), salmon (s) and chicken II (cII)-type GnRHs and various pituitary hormones. Both sGnRH and mGnRH appeared to be colocalized in the same cells of the nervus terminalis. Chicken GnRH II-immunoreactivity was found only in fibers and terminals. In the younger fish [73 and 186 days after fertilization (DAF)] GnRH neurons were seen rostral to the olfactory bulb. A novel GnRH ganglion, along the nervus terminalis, was found at the cribiform bone (gCB). A few non-immunoreactive rounded cells were seen among the GnRH neurons. A second smaller ganglion was seen at the most rostrally located part of the ventromedial olfactory bulb (gROB). In the older fish (850 DAF) GnRH neurons were also observed in the basal forebrain. A small group of neurons (2-3 cells), at the caudoventromedial border of the olfactory bulb, formed the ganglion terminale. Occasionally isolated GnRH-immunoreactive cells were seen at the base of the olfactory epithelium, along the ventromedial margins of the olfactory nerve. GnRH-immunoreactive and GnRH mRNA expressing neurons were absent from midbrain regions at the ages observed. GnRH-immunoreactive fibers were present only in older fish. The pattern of distribution of fibers that were immunoreactive to all three forms of GnRH was identical. Fibers were seen along the medial side of the olfactory nerve, throughout the brain and in the pituitary, associated with growth hormone and somatolactin cells.(ABSTRACT TRUNCATED AT 250 WORDS)

To determine the possible involvement of reactive oxygen species in ovulation, dynamic aspects of superoxide dismutase (SOD) isozyme were studied in the ovaries of rats by in situ hybridization histochemistry. Previously, mRNA levels of ovarian manganese superoxide dismutase (Mn-SOD) were reported markedly to increase whilst enzymic activity of Mn-SOD decreased during the ovulatory process after treating immature rats with 10 and 5 Units, respectively, of pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG). Levels of Cu/Zn-SOD activity and Cu/Zn-SOD mRNA were reported to remain unchanged throughout ovulation. This increase in the Mn-SOD mRNA level was shown in the present study by in situ hybridization to be localized to the theca interna cells throughout the PMSG/HCG-induced ovulatory process. The observations suggest that the turnover rate of Mn-SOD but not Cu/Zn-SOD increases specifically in the mitochondria of these cells. SOD has been postulated to play important roles in steroidogenesis. The relationship is discussed between mitochondrial functions in steroid-secreting cells and superoxide radicals and related metabolite(s).

Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca(2+)-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

To measure quantitatively the intracellular distribution of cellular glutathione peroxidase (GPX) in rat hepatocytes, ultrathin sections were stained by a postembedding immunogold technique. GPX had a specific activity of 1670 Units/mg protein, and was purified 2050-fold from rat liver by means of heat denaturation, ammonium sulfate fractionation, and a series of chromatographic procedures including thiol-Sepharose 4B. The purified GPX was shown to be electrophoretically pure, and was a homotetramer of 22 kDa subunits. Monospecific polyclonal antibodies were raised in rabbits by immunization. By immunoblot analysis, both the light mitochondrial the and cytosolic fractions of rat liver homogenate gave a single band with an identical mobility to that of the purified enzyme. Under the light microscope, hepatocytes showed nuclear staining and granular cytoplasmic staining, corresponding to certain intracellular structures. The labeling density (number of gold particles/microns 2) for GPX obtained by immunoelectron microscopy was 11.9 in the nuclei, 19.6 in mitochondria, 3.32 in peroxisomes, 1.95 in lysosomes, and 9.81 in the cytoplasmic matrix. These results suggest that cellular GPX is present in various compartments of rat hepatocytes, and that the GPX occurs in relatively higher amounts in mitochondria.

Feasibility of the combination of the immunogold-silver staining method (IGSS) and computer-aided image analysis was assessed for the detection of antigen in an immunostained, paraffin-embedded section. Using low-temperature IGSS, we stained a specimen of human oral squamous cell carcinoma with a monoclonal antibody, PC 10, against a proliferating cell nuclear antigen (PCNA/cyclin), and the section was analyzed by ACAS 570 interactive laser cytometry. The PCNA-positive cells, exhibiting a heteromorphic texture, were contrasted by the dark staining of their nuclei, but showed heterogeneity in staining intensity from cell to cell. Using a conventional microscope light source rather than a laser, and by employing the COMPLEMENT DATA program (which permits inversion of the data values) installed in the ACAS 570 software system, we were able to obtain a 'complemental image' which replicated the real immunohisto-morphology. Approximately 30-35 cells from three different areas in the same section were selected by DEFINE CELL and MARK AREA programs, and quantitative image analysis was performed in terms of cell integrated value, area, perimeter, and shape factor indicated in histogram form. The combined utilization of IGSS with computer-aided image analysis was demonstrated to offer a crucial advantage for the quantitative assessment of immunostained sections.