Showing papers in "Histochemistry and Cell Biology in 1992"

Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O.

Abstract: Cultured 3T3-F442A cells differentiate into adipocytes and accumulate lipid droplets in the cytoplasm. When fat cells are stained with Oil red O, the degree of staining seems to be proportional to the extent of cell differentiation. We report here a fast and simple method to quantitate the extent of adipose conversion by staining the accumulated lipid with Oil red O and determining the amount of extracted dye at 510 nm. The results show that Oil red O specifically stains triglycerides and cholesteryl oleate but no other lipids. This technique is a valuable tool for processing large numbers of cell cultures or samples in which adipose differentiation and/or accumulated triglycerides is to be quantitated.

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Abstract: In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine.

Abstract: Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with NG-nitro-L-arginine, a NOS inhibitor, or L-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms.

Abstract: Nitric oxide (NO) is generated from l-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Antisera A and B reacted with a ∼ 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of Mr between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the ∼ 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord. Therefore, antibodies to rat brain enzyme react with the human equivalent and also with other NO synthase isoforms in human endothelium. These findings support the contention that the endothelial enzyme is a different form with partial homology to that in nerves and also provide an anatomical distribution of NO synthase isoforms.

Distribution of hyaluronan and its CD44 receptor in the epithelia of human skin appendages

Abstract: Biotinylated hyaluronan (HA) binding complex (HABC) from bovine articular cartilage proteoglycan was used as a histological probe to study the localization of HA in human skin. The distribution of HA was compared with its presumptive cell surface receptor, CD44, using monoclonal antibodies. In epidermis both HA and CD44 were found in the basal and spinous cell layers, but neither was present in the stratum granulosum and stratum corneum. In the keratinizing parts of hair follicles, i.e. in the outer and inner epidermal root sheath, pilosebaceous duct and the actual hair, HA and CD44 were found between the vital but not the terminally differentiated cells. In the sebaceous glands a small amount of HA was found around all cells, whereas CD44 was restricted to the basal cell layer. The secretory acini of the sweat glands stained intensively with anti-CD44 antibodies but only weakly with HABC. In the sweat gland, CD44 was localized on the basal and lateral surfaces of the clear cells, whereas the dark cells and the myoepithelial cells were negative. Both the lower and upper layers of the sweat gland ducts showed a faint but constant staining for CD44 and only minor amounts of HA. While in the keratinizing skin epithelia both HA and its CD44 receptor showed an intense staining with a close co-distribution, in the sweat and sebaceous glands their distribution patterns were not similar. It is suggested that in epithelia with divergent differentiation programs the functions of CD44 and HA may be different.

Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.

Abstract: The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3′ end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

Which fluorophore is brightest? A comparison of the staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, texas red, and cyanine 3.18

Abstract: There are several red-emitting fluorophores available for immunofluorescence studies, including tetramethylrhodamine, lissamine rhodamine, Texas Red, and cyanine 3.18; however, it is unclear which of these is best. The present study compared the brightness of these fluorophores to that of fluorescein. Staining was attempted using a primary antibody raised against serotonin and a secondary antibody that was conjugated with either fluorescein or one of the red fluorophores. The intensity of staining was determined densitometrically. It was found that a conjugate of cyanine 3.18 provided significantly brighter staining that conjugates of any of the other fluorophores, including fluorescein. It is concluded that cyanine 3.18 should be useful for multicolor fluorescence experiments and that it may be the brightest fluorophore available for single-color fluorescence immunocytochemistry.

Nile red staining of lysosomal phospholipid inclusions.

Abstract: We have employed the fluorescent dye nile red to distinguish between normal cells and cells containing lysosomal accumulations of phospholipids. When fibroblasts from an individual with a genetic deficiency in lysosomal sphingomyelinase activity (Niemann-Pick disease) were stained with nile red and visualized by fluorescence microscopy, orange-colored inclusions were observed throughout the cytoplasm. The orange fluorescent bodies could be distinguished from the neutral lipid droplets that fluoresce a brilliant yellow-gold in the presence of nile red. These inclusions were also observed in alveolar macrophages obtained from rats treated with amiodarone, an antiarrhythmic agent known to produce lysosomal phospholipidosis. Flow cytofluorometric analysis revealed that staining of these phospholipid-rich macrophages with nile red can distinguish them from control alveolar macrophages. These results demonstrate that nile red can be employed for the rapid staining of cellular phospholipid inclusions.

Identification of brush cells in the alimentary and respiratory system by antibodies to villin and fimbrin.

Abstract: Brush cells represent a population of epithelial cells with unknown function, which are scattered throughout the epithelial lining of both the respiratory system and the alimentary system. These cells are reliably distinguished from other epithelial cells only at the ultrastructural level by the presence of an apical tuft of stiff microvilli and extremely long microvillar rootlets that may project down to the perinuclear space. In the present study we show that brush cells can be identified in tissue sections even at the light microscopic level by immunostaining with antibodies against villin and fimbrin, two proteins that crosslink actin filaments to form bundles. In brush cells, villin and fimbrin are not only present in the actin filament core bundles of apical microvilli and their long rootlets but, in addition, both proteins are also associated with microvilli extending from the basolateral cell surface of the brush cells. Basolateral immunostaining specific for villin and fimbrin does not occur in any other epithelial cell type of the respiratory and alimentary tract. Thus immunostaining with antibodies against both proteins allows unequivocal identification of individual brush cells even in sectional planes that do not contain the brightly stained apical tuft of microvilli and their long rootlets.

Distribution of sugar residues in the bovine testis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates.

Abstract: In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to α-d-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. α-d-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal d-Gal-(β1→3)-d-GalNAc, d-Gal and d-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited d-Gal, d-GlcNAc or sialic acid and d-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (α-d-Man), RCA-I (d-Gal), UEA-I (α-l-Fuc) and WGA (d-GlcNAc or sialic acid).

Calcitonin gene-related peptide, substance P and GAP-43/B-50 immunoreactivity in the normal and arthrotic knee joint of the mouse.

Abstract: The aim of this study was to describe the normal distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) containing fibres in the knee joint of the mouse and to obtain insight into the changes in innervation associated with degenerative processes in the joint. Arthrosis was induced by a single subpatellar intra-articular injection of bacterial collagenase. After decalcification in EDTA solutions, the CGRP and SP fibres were visualized by peroxidase-antiperoxidase pre-embedding immunocytochemistry for light microscopy. Control experiments on the mouse brain as a reference for the effect of EDTA on the immunostaining showed that the decalcification procedure with EDTA had not impaired the immunostaining. A rich innervation of thin varicose CGRP and SP immunoreactive fibres was found in most peri- and intra-articular tissue components. The periosteum, synovial tissues, the joint capsule and the intra-articular fat tissues were richly innervated. Less intense innervations were also found in the subchondral bone plates of the tibio-femoral joint and of the patella. Fibres were also found in the soft tissues between the patellar tendon and the femoral groove. No differences could be found between the location of CGRP and SP fibres with respect to the localization in the joint, but generally more CGRP fibres were found. The collagenase-induced osteoarthrosis was characterized by sclerosis of the subchondral bone, patellar dislocation, osteophyte formation, synovial proliferation and by severe cartilage abrasion, particularly on the medial side of the femoro-tibial joint. The overall distribution of CGRP and SP fibres was the same as in the control joints. However, major differences were found in all studied joints at specific locations around the cruciate ligaments, in the synovium around the patella, in the soft tissues lateral of the patella and in plica tissue between the patella and femoral groove. The CGRP and SP innervation was no longer detectable by immunolabelling with the antibodies. With a polyclonal antibody to the growth associated protein GAP-43/B-50, signs of degenerated axonal profiles were observed in these locations. At other peripheral locations, such as the muscles, the GAP-43/B-50 distribution was normal. In conclusion, the present study provides detailed information on the localization of CGRP and SP fibres, which may be involved in pain perception. Knowledge of the changes that occur during arthrosis may give more insight into the clinical symptoms.

Labeling with fluorescent carbocyanine dyes of cultured endothelial and smooth muscle cells by growth in dye-containing medium.

Abstract: We describe a method for labeling cultured endothelial cells (ECs) and smooth muscle cells (SMCs) by letting the cells grow for three days in culture medium containing a low concentration of the fluorescent carbocyanine dyes DiI and DiO. We show that good labeling can be obtained with considerably lower concentrations (2.5 μg/ml) than has previously been described. With optimal concentration the labeling is very strong and seems to label all membranous structures in the cells. It was possible to clearly distinguish differentially pre-labeled cells both in coculture and seeded on denaturated vascular grafts. The cells remain fluorescent for more than seven days and may be passaged with retained proliferative capability. We suggest that DiI/DiO-labeling using dye-containing medium may be used for several cell types and is applicable in tissue culture and in the detection of implanted cells in vivo.

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

Abstract: The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vescicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.

Comparative lectin histochemistry on taste buds in foliate, circumvallate and fungiform papillae of the rabbit tongue.

Abstract: Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungiform taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like, paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.

Localization of collagen X in human fetal and juvenile articular cartilage and bone.

Abstract: The tissue localization was analysed of collagen X during human fetal and juvenile articular cartilagebone metamorphosis. This unique collagen type was found in the hypertrophic cartilage zone peri- and extracellularly and in cartilage residues within bone trabeculae. In addition, occasionally a slight intracellular staining reaction was found in prehypertrophic proliferating chondrocytes and in chondrocytes surrounding vascular channels. A slight staining was also seen in the zone of periosteal ossification and occasionally at the transition zone of the perichondrium to resting cartilage. Our data provide evidence that the appearance of collagen X is mainly associated with cartilage hypertrophy, analogous to the reported tissue distribution of this collagen type in animals. In addition, we observed an increased and often “spotty” distribution of collagen X with increasing cartilage “degeneration” associated with the closure of the growth plate. In basal hypertrophic cartilage areas, a co-distribution of collagens II and X was found with very little and “spotty” collagen III. In juvenile cartilage areas around single hypertrophic chondrocytes, co-localization of collagens X and I was also detected.

Effect of traumatic occlusion on cgrp and sp immunoreactive nerve fibre morphology in rat molar pulp and periodontium

Abstract: Traumatic occlusion provides a trauma that affects the whole tooth and its supporting tissues. To study the effect of this trauma on CGRP and SP immunoreactive nerve morphology in pulp and periodontium, traumatic occlusion was induced in 2-months-old rats. The occlusal surface of the first maxillary molar in 30 rats were unilaterally raised 1 mm with a composite material. At different observation periods up to 30 days, the rats were transcardiacally perfused, the jaws demineralized, sectioned and processed for immunohistochemistry with the avidin-biotin-peroxidase method. Changes in nerve morphology, distribution and density in first and second molars and their supporting tissues were analyzed and compared in experimental (n=30) and control rats (n=14). Already after 5 days with traumatic occlusion, 22% of the experimental teeth had increased density of CGRP and SP immunoreactive nerves locally in gingiva, the periodontal ligament and the pulp, while in 15%, axonal proliferation and changed nerve morphology were found in the whole pulp (severe reaction). During a 20-day period, the pulpal nerve reactions progressed and included the whole pulp in 46% of the experimental teeth. The periodontal nerve responses were still localized only to the cervical and apical regions, and they remained local in these areas throughout the experimental periods. After 20 days the number of teeth with severe nerve changes seemed to decrease. The study shows that an unilateral change in occlusion of the first molar initiate nerve responses in the total molar dentition. In this experimental model the pulpal axons containing CGRP and SP reacted more serious to occlusal trauma than the nerves in the periodontium. The results indicate that the nerve changes in some cases might be transient.

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method.

Abstract: The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl2 to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl2 and CeCl3. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.

Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine.

Abstract: The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of lfuorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.

Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections.

Abstract: Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehydefixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.

Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation.

Abstract: The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.

Nuclear receptor sites for vitamin D-soltriol in midbrain and hindbrain of Siberian hamster (Phodopus sungorus) assessed by autoradiography.

Abstract: Autoradiograms were prepared from midbrains and hindbrains of male and female Siberian hamsters (Phodopus sungorus), kept under short-day or long-day illumination, after injection of tritium-labeled 1,25-dihydroxycholecalciferol (vitamin D, soltriol). Concentration and retention of radioactivity was noted in nuclei of certain neurons, glial cells, and ependymal cells, and in choroid epithelium. Labeled neurons of varying intensity were found throughout the brainstem in distinct populations at characteristic topographical sites, which include cranial nerve motor nuclei, the nucleus (n.) reticularis tegmenti pontis, the caudoventral region of the n. raphe dorsalis, the n. trapezoides, the n. vestibularis lateralis and n. vestibularis superior, neurons in the various nuclei of the sensory trigeminus, accessory optic nuclei, scattered neurons in nuclei of the reticular formation, the n. ambiguus, certain cells in the area postrema, and many others. Glial cells with nuclear labeling, probably microglia, were scattered predominantly in or near myelinated nerve fascicles. The choroid epithelium showed strong nuclear labeling throughout the ventricle. Nuclear labeling of ependyma was variable and weak, mainly at ventral and lateral extensions (recesses) of the ventricle. The extensive presence of nuclear binding in select neural structures indicates that vitamin D exerts specific genomic effects on cell populations that are known to be involved in the regulation of motor, sensory, autonomic, neuroendocrine, metabolic, and immune functions. The results of these studies, in conjunction with those from other brain and peripheral tissues, recognize vitamin D-soltriol as a steroid hormone with a wide scope of hormone-specific target cells, similar to estrogen, androgen, and adrenal steroids, and which are topographically distinct and characteristic for its functions as the steroid hormone of sunlight.

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies.

Abstract: A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.

Immunocytochemical localization of cathepsin D in the rat osteoclast.

Abstract: We performed immunocytochemical localization of cathepsin D in osteoclasts of the proximal growth plate of the rat femurs using both the avidin-biotin-peroxidase complex method for cryo-semi-thin (1 μm) sections and the colloidal gold-labeled IgG method for K4M ultra-thin sections. At the light microscopic level, cathepsin D immunoreactivity in the osteoclasts appeared at the vesicles, granules, and/or small vacuoles. They were distributed throughout the cytoplasm of each cell and were relatively numerous close to the bone surface. This antigen could not be detected at the eroded bone surface. As for other cells, immunoreactivity was seen only in the lysosomes of osteoblast-like cells. Immunoreactivity in the osteoclasts was stronger and greater in the density and number than in osteoblast-like cells. At the electron microscopic level, osteoclasts with well-developed ruffled border possessed numerous cathepsin D-containing lysosomes, vacuoles, and coated vesicle-like structures. Cathepsin D-containing lysosomes fused with cathepsinnegative vacuoles and formed large secondary lysosomes. Osteoclasts with poorly developed ruffled border possessed fewer cathepsin D-containing lysosomes than those with well-developed ruffled border. No immunogold particles were seen in vacuole-like channel expansions of the ruffled borders, between the channels of the ruffled borders, or on the eroded bone surface. These findings demonstrate that osteoclasts contain a large amount of cathepsin D. They suggest that cathepsin D is necessary for osteoclastic bone resorption, that it plays an indirect rather than direct role.

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney

Abstract: In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.

Immunocytochemical localization of a galanin-like peptidergic system in the brain of two urodele and two anuran species (Amphibia).

Abstract: Galanin-like immunoreactivity was localized in the brain of Urodela (Ambystoma, Pleurodeles) and Anura (Bufo, Xenopus) by immunocytochemistry with anti-porcine galanin antiserum. In the four species, immunoreactive perikarya were observed in the telencephalon (striatum, amygdala), diencephalon preoptic area mainly along the anterodorsal wall of the preoptic recessus, suprachiasmatic nucleus, lateral hypothalamus, ventral and dorsal infundibular nuclei, paraventricular organ, and rhombencephalon (nucleus of the solitary tract). Galaninergic fibres extended in similar regions and in the medial septum, ventral telencephalon, ventral hypothalamus, median eminence, and various mesencephalic and rhombencephalic regions. Contacts with the cerebrospinal fluid cavity occurred along the preoptic recessus (Ambystoma) and the ventral infundibular wall (all species). Fibres were scarce in the neurohypophysis. The distal and intermediate lobes of the pituitary were virtually devoid of immunoreactivity. The galaninergic system appeared more developed in adult amphibia than in young animals, suggesting the stimulating influence of sex steroids on the expression of galanin as previously described inAnguilla. The extensive distribution of the galanin-like immunoreactive neurons in amphibian brains suggests that this peptide may act as a neuromodulatur and/or neurotransmitter.

Vimentin antibodies stain membranous epithelial cells in the rabbit bronchus-associated lymphoid tissue (BALT)

Abstract: The lymphoepithelium covering the bronchus-associated lymphoid tissue (BALT) of the rabbit lung was studied with monoclonal antibodies against vimentin, using the indirect immunoperoxidase technique. In the lymphoepithelium single cells which had a membranous apical cytoplasm and engulfed intraepithelial lymphocytes were vimentin-immunoreactive. All other epithelial cells of the lymphoepithelium and of the surrounding airway epithelium did not bind vimentin antibodies. The results support the hypothesis that the membranous epithelial cells in the lymphoepithelium of rabbit BALT are analogous with intestinal M-cells, which in rabbit Peyer's patches and appendix are selectively labelled by vimentin antibodies.

Enzyme and immunohistochemical assessment of myocardial damage after ischaemia and reperfusion in a closed-chest pig model.

Abstract: The usefulness of different enzyme and immunohistochemical stains to distinguish reversible and irreversible myocardial cell injury after experimental coronary artery occlusion of varying duration and reperfusion with or without superoxide dismutase as adjunct was investigated Biopsies or parts of the infarcted and non-infarcted area were rapidly frozen and sectioned in series for enzyme and immunohistochemical evaluation Sections were stained for the demonstration of phosphorylase, myofibrillar ATPase and mitochondrial oxidative enzymes and also with periodic acid-Schiff, alizarin red S and routine histological stains Other sections in series were stained with antibodies against fibronectin and the intermediate filament proteins desmin and vimentin In 49 biopsies a blind quantitative estimation of the area stained for fibronectin, phosphorylase and alizarin red S was performed and evaluated statistically Phosphorylase, periodic acid-Schiff, fibronectin and alizarin red S allowed delineation of affected myocardium after 30 min of ischaemia followed by reperfusion whereas with the other stains, affected myocardium was readily detectable only after 60 or 90 min of ischaemia followed by reperfusion as well as after 24 h of ischaemia without reperfusion The immunostaining for fibronectin was very distinct and inversely related to the phosphorylase activity We show that fibronectin is an excellent marker for damaged cells and that these positively stained myocytes are necrotic as confirmed ultrastructurally Using alizarin red S as a marker of calcium accumulation in myocytes, a marked discrepancy was observed between the area of fibronectin-containing myocytes and that of myocytes stained by alizarin red S Calcium accumulation in mitochondria is thus not a prerequisite for myocyte necrosis but does occur only in some of the irreversibly damaged cells Of special interest is the finding that there was a significant reduction of intracellular calcium in pigs where superoxide dismutase had been used as an adjunct at reperfusion, thus supporting the theory that free radicals do play a role during reperfusion of ischaemic myocardium

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes.

Abstract: The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.

Distribution of estrogen and progesteron receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit.

Abstract: In order to clarify the distribution and content of estrogen (ER) and progesteron receptors (PR) under changing hormonal influences within the various cell populations of the uterus (glandular and luminal endometrial epithelium, stroma, myometrium), immunohistochemical determinations using specific monoclonal antibodies were made. To correlate the immunohistochemical findings with peripheral hormone levels and specific tasks of the endometrium, 17β-estradiol and progesterone serum levels were measured and cell proliferation determined by use of BrdU-labelling-immunohistochemistry. At the subcellular level ER and PR were located exclusively in the cell nuclei of female rabbits, which were either immature and lacking any peripheral hormone levels or were pseudopregnant (d0–d8 p.hCG). In the immature rabbits a general faint ER and PR immunostaining was found. In addition to a general increase in ER and PR in all cell populations estrous rabbits (d0 p.hCG) showed a significant rise of ER in the epithelial cells and of PR in the myometrium. Within the epithelial cells and the myometrium the ER dropped heavily within a few days of pseudopregnancy. The PR, however, increased sharply during the first two days of pseudopregnancy and decreased gradually following d4 p.hCG. A close relationship was observed between the high PR content and the proliferation rate of the epithelial cells on d2 p.hCG. In spite of the more rapid decrease of ER compared with PR, the glandular epithelium retained positive immunostaining. In the stroma the ER and especially PR content did not change significantly during the course of pseudopregnancy suggesting that some of the well-known differentiation events in the luminal epithelium may be mediated by the stroma.

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Abstract: Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.