Staining Of Ultrathin Sections Research Articles

The role of ultrastructural abnormalities of the blood-brain barrier in the development of brain glioblastoma radioresistance

Background: Glioblastoma (GB) is the most commonly diagnosed brain tumor. Its management involves adjuvant therapies, such as radiation. The cause of high probability of GB local relapse is its radioresistance related to hypoxia arising from abnormal blood-brain barrier permeability in GB vessels and in the peritumoral zone (PZ). Aim: To study pathophysiology of hypoxia in the residual GB based on the abnormalities of the morphological elements of the capillary walls building up the blood-brain barrier in GB and PZ capillaries. Materials and methods: Samples for morphological evaluation were taken during surgery for GB in 5 patients. The samples were prepared for transmission electron microscopy according to the standard technique with fixation in 2% glutaraldehyde in phosphate buffer, post-fixation with osmium tetroxide, embedding in the epon-araldite mixture, and contrast staining of ultrathin sections with uranylacetate and lead citrate. Abnormalities of the capillary cells (mitochondrial vacuolization and vacuolization of endoplasmic reticulum in endothelial cells, pericytes and astrocytes), as well as of the acellular element of the capillary wall, i.e. basement membrane, were assessed in two groups of capillaries – those of GB (n = 38) and those of PZ (n = 32). Results: Abnormalities characteristic for apoptosis and oncosis were found in the cells of the GB and PZ capillaries of the blood-brain barrier, such as endothelial cells and pericytes. However, in the GB capillaries these abnormalities were signifcantly more frequent (р < 0.001). Only half (52.6%) of the GB capillaries had an edematous pericapillary astrocyte layer. In all other capillaries, astrocyte sprouts either were visualized as separate morphological elements (13.2%) or were not visualized at all (34.2%). All PZ capillaries had the astrocyte layer, being edematous in 68.8% of the capillaries and totally edematous only in 25%. Thickened basement membrane was found in the vast majority (89.5%) of the GB capillaries and only in 25% of the PZ capillaries (р < 0.001). Conclusion: Findings of abnormal cell elements in the GB capillaries leading to peritumoral edema and consequent hypoxia are highly likely to be the cause of the remnant GB radioresistance.

Contrast Enhancement with Oolong Tea in Staining of Ultrathin Sections

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Synaptic organization of substance P, glutamate and GABA-immunoreactive boutons on functionally identified neurons in cat spinal deeper dorsal horn

In order to determine how nociceptive input conveyed by the C-fibers terminating in superficial laminae of the spinal cord reaches the wide dynamic range (WDR) cells in deeper dorsal horn, which functions as ascending projection pathway, the morphological features of some WDR cells in the deeper dorsal horn of the cat lumbar spinal cord were studied by intracellular injection of horseradish peroxidase and physiological characterization. One of the fully stained neurons with somata in lamina V and dendrites that entered lamina II were examined by electron microscopy. Immunogold staining of ultrathin sections through the labeled proximal dendrites in lamina II revealed that these dendrites received numerous synapses from substance P and glutamate immunoreactive (IR) axons, which were considered originating from C-fibers. In addition, many GABA-IR terminals were found presynaptic to the labeled dendrites. The results, therefore, suggest that the information carried by primary afferent can be sent from the superficial dorsal horn to the deeper laminae through monosynaptic contacts between C-fiber terminals and the long dorsal dendrites of some WDR cells in the deeper laminae, and that GABAergic system is involved in postsynaptic control to modulate the transmission of nociceptive sensory information.

Visualization of activation-dependent epitopes on glycoprotein IIb-IIIa complexes of platelets stimulated by thrombin: immunogold staining of ultrathin sections.

An immunoglobulin M monoclonal antibody (IgM MAb; AP-6) recognizing the sequence 211-221 of glycoprotein (GP) IIIa enabled a study of the distribution of this epitope on unstimulated or thrombin-activated platelets. Flow cytometry was used to evaluate the expression of this epitope on platelets and immunogold staining on ultrathin sections to analyze its distribution within the cell. There was little or no binding of AP-6 to unstimulated platelets, but immunogold staining showed labeling associated with the membranes of alpha-granules. The binding of AP-6 to thrombin-stimulated platelets was compared with that of anti-RIBS MAbs and antifibrinogen polyclonal antibodies. An increased expression of the AP6 epitope was observed on membranes of the surface-connected canalicular system and at the periphery of the cell as early as 10 to 15 seconds after platelet activation by thrombin. At the same time, binding of anti-RIBS MAbs confirmed that at least part of the endogenous fibrinogen had left the alpha-granules and was bound to platelet membranes. Staining with polyclonal antifibrinogen antibody also revealed fibrinogen in vesicles resulting from granule fusion. Rapidly, the pool of internal membranes with bound fibrinogen became exposed to the outside of the platelet, a process involving the unfolding of membranes and pseudopod formation. Our results provide evidence that activation of GP IIb-IIIa and binding of fibrinogen to platelet membranes can occur before their expression at the platelet surface. They also suggest the presence of an alpha-granule pool of GP IIb-IIIa linked to fibrinogen in unstimulated platelets.

The physical characteristics of anaerobic granular sludges in relation to their internal architecture

A range of granular sludges was taken from industrial anaerobic sludge blanket reactors treating a wide variety of wastewaters and a comparison was made between the polymers which were extractable from the granules and their internal structures. The study of the internal structure, using sequential staining of ultra-thin sections, showed the complexity of granular sludges. Much of the area was occupied by Gram-negative cells and the area which stained positive for protein was found to increase nearer the centre of the granules. This was accompanied by a decrease in the carbohydrate positive areas. Positive areas for lipid were widespread throughout the granules. Changes in the internal structure were observed when the type of wastewater treated by the granules was changed and a comparison between sludges treating the same type of wastewater showed that factors other than the nature of the substrate must be considered as parameters which will affect the structure of the granules. Although an appreciable variation in the granule strengths was noted, it was not possible to relate these differences, on an overall basis, to either the internal structure or the chemical composition of the extracted polymers. However, an examination of data for granules produced during the treatment of nominally similar wastes did suggest that there would be a relationship between polymer composition and granule strength in these cases.

Three-Dimensional reconstruction of protein bodies in developing maize endosperm using immunocytochemical techniques

The storage proteins of maize (Zea mays L.), like many other cereals, are a group of alcohol-soluble proteins classified as prolamines. In maize these proteins comprise four structurally distinct types and are termed zeins. Zein synthesis is initiated in developing maize endosperm and continues until the seed reaches maturity (between approximately 12 to 50 days after pollination). Zeins are synthesized by polysomes bound to the rough endoplasmic reticulum (RER) and they accumulate as insoluble aggregates called protein bodies. Our previous studies have shown that the zeins are deposited within protein bodies in a defined order in normal genotypes. However, the actual three-dimensional organization of the zeins within protein bodies and the interactions that occur between the various zeins is not known. Our goal is to understand protein body organization and the interactions that occur between the various proteins.The four types of zeins are identified by their molecular weight after separation by SDS-PAGE, and are designated α-, β-, γ- and 6-zeins. The β-, γ-, and δ-zeins are all sulfur-rich proteins, while the α-zeins contain only low amounts of cysteine and methionine. These proteins can be individually detected by immunocytochemical staining of ultrathin sections. Additionally, ultrathin sections poststained with lead citrate and uranyl acetate demonstrate that there are two distinct regions that correlate with the immunocytochemical staining patterns.

Appearance of myofibroblast during the healing of experimental corneal wound

It is well known that corneal stroma cells (keratocytes) can transform to fibroblasts during the corneal wound healing. We have studied the expression of α-smooth muscle actin, vimentin and desmin in fibroblastic cells of the alkali-burneo and lacerated corneas in the rabbits by means of immunohistochemistry and electron microscopy.Methods. Rabbits were anesthetized, and central corneal alkali-burn and laceration were produced. The injured corneas healed for 1 day to 45 days, and 18 days embryonic rabbit corneas were immunostained with monoclonal antibodies against α-smooth muscle actin, vimentin and desmin. For transmission electron microscopy, two techniques were used for the staining of ultra-thin sections: 1) uranyl acetate and Reynold's lead citrate and 2) the tannic acid method of Kajikawa.Results. Antibody against α-smooth muscle actin reacted with fibroblastic cells in injured corneas (Fig 1) but not with stromal cells in normal and embryonic corneas. Antivimentin antibody strongly reacted with the fibroblastic cells in injured corneas (Fig 2) and stromal cells in normal and embryonic corneas.

Incubation chambers for the staining of ultrathin sections. An in situ technique especially suited for immunocytochemistry.

The preparation and use of incubation chambers for staining of ultrathin sections is described. As an example the immunogold staining is given which can be carried out in these devices in a more simple manner than normally possible, and with good results as shown by electron microscopy.

Interferon‐gamma gene and protein are spontaneously expressed by the porcine trophectoderm early in gestation

The nature and the source of the antiviral activity found in the reproductive tract of pregnant gilts early in gestation were analyzed. Two antigenically distinct antiviral activities were found in uterine flushings and in supernatants of conceptus-conditioned culture medium between days 12 and 20 of gestation, using Madin Darby bovine kidney cells and vesicular stomatitis virus as a challenge in the antiviral bioassay. One component was antigenically identified as interferon-gamma (IFN-gamma). Northern blot analysis of conceptus poly(A)+ RNA with a human IFN-gamma cDNA probe revealed two mRNA of 1.3 and 1.4 kb. In addition, immunoprecipitation of metabolically labeled conceptus secretory proteins with an antiserum raised against purified porcine rIFN-gamma resulted in four bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular mass 18.5 to 24.5 kDa. Pre-electrophoresis incubation of the immunoprecipitate with glycopeptidase F, which removes N-linked carbohydrates, yielded a single band of 16.5 kDa. Finally, staining of ultrathin sections by indirect immunofluorescence using the same antiserum to rIFN-gamma revealed that all cells of extra-embryonic trophectoderm contained intensely fluorescent granules in their apical cytoplasm. Neither endoderm nor embryonic cells stained positive. These results clearly show that IFN-gamma, known so far as a T or NK cell-derived lymphokine, is spontaneously and intensively secreted by the porcine trophectoderm, an embryonic tissue not related to the hematopoietic lineage. They also suggest that the implanting conceptus, at least in the porcine species, could play an active role in immune interactions with the mother.

Ultrastructure and cytochemistry of squamulae intravaginales of the marine angiosperm, Halophila ovalis

Small delicate structures found in the leaf bases in vegetative buds of the marine angiosperm Halophila ovalis (R. Br.) Hook. f. showed a fine structure of the component cells typical of secretory cells. They are rich in organelles particularly ER, dictyosomes, ribosomes and mitochondria. Endoplasmic reticulum occurs at two localities in each cell, one surrounding the central nucleus and the other in the peripheral regions close to the plasmalemma. Groups of dictyosomes occur between the two layers of ER and each group is separated from another by interconnecting strands of ER. A mucilaginous secretion accumulates on the cell surface and between the outer and inner layers of cell wall, particularly at the corners. The involvement of ER in the secretion of mucilage is discussed. Staining of ultrathin sections with silver proteinate after thiocarbohydrazide showed the presence of insoluble carbohydrates and histochemical tests with alcian dyes showed the secretion to contain non-sulphated acid polysaccarides. Staining of epoxy resin sections with Coomassie blue R250 indicated that the mucilage secretion on the surface of and within the cell walls also contained proteins. Cellulase–gold complex marked the cellulose wall layers as distinct from the included secretion. Klein, delikate strukture wat by die blaarbasisse in vegetatiewe knoppe van die marine angiosperm Halophila ovalis (R. Br.) Hook. f. gevind is, het ’n fyn struktuur van saamgestelde tipiese afskeiselle getoon. Hulle is ryk aan organelle, veral endoplasmiese retikulum (ER), diktiosome, ribosome en mitochondria. ER het op twee plekke in elke sel voorgekom, romdom die sentrale kern asook in die periferale dele na aan die plasmalemma. Groepe diktiosome kom tussen die twee lae van ER voor en elke groep is van die ander geskei deur aaneengeskakelde stringe van ER. Slymerige afskeiding versamel op die seloppervlak en tussen die binneste en buitenste selwandlae, veral in die hoeke. Die betrokkenheid van ER in die afskeiding van slym word bespreek. Kleuring van ultradun snitte met tiokarbohidrasied gevolg deur silwer-proteïnaat het getoon dat onoplosbare karbonate teenwoordig is en histochemiese toetse met alcian-kleurstowwe het aangetoon dat die afskeiding nie-sulfaat-suur polisakkariedes bevat. Kleuring van epoksiharsseksies met Koomassie-blou R250 het aangedui dat die slymafskeiding op die oppervlak en ook binne die selwande proteïne bevat. Behandeling met ’n sellulase-goudpeilstaafhet die sellulose selwande van die sekresie onderskei.

A Malignant Tumor of the Pancreas Producing Glucagonoma Syndrome

In this study, liver metastases from a patient with a pancreatic glucagonoma producing the syndrome have been investigated histologically, ultrastructurally, and immunocytochemically. A comparison has also been made between the metastases and the primary pancreatic tumor investigated in a parallel study. In the metastatic tissue, glucagon-, pancreatic polypeptide (PP)-, and somatostatin-containing cells were found together with a majority of cells without any immunoreactivity. Glucagon-positive cells were much more numerous than PP- and somatostatin-immunoreactive cells. As in the primary tumor, double immunogold staining of ultrathin sections demonstrated the co-existence of glucagon and PP immunoreactivities in most of the granulated cells, but PP immunolabeling was often faint, so that it probably could not be revealed by the PAP method in light microscopical sections. Such a finding, together with the histological and ultrastructural features, is consistent with an ontogenic and phylogenetic primitiveness of the metastatic cell population.

Experimental Studies of the Elastic Fiber of the Capsular Ligament: Influence of Ageing and Sex Hormones on the Hip Joint Capsule of Rats

Physiological changes with advancing age and the influence of sex hormones on elastin in the hip joint capsule of rats were studied both biochemically and morphologically. Elastin content of the joint capsule was greater in females than in males at 10 weeks of age. Staining of ultrathin sections with Verhoeff iron hematoxylin followed by lead citrate (VIH-LC) demonstrated that the elastic fibers of specimens from infant rats were characterized by a small elastin core formed by aggregation of elastin fibrils in the center of closely packed microfibrils. With advancing age the diameter of the elastic fiber of the hip joint capsule tended to increase gradually in both sexes until sexual maturation was reached. In specimens from adult rats, the elastic fiber consisted of an increased solid elastin core surrounded by loosely packed microfibrils. The diameter of the elastic fiber and the ratio of the central elastin to the peripheral microfibrils were greater in females than in males after sexual maturation. Th...

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied; After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver straining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.

Improved staining boxes for fast, uniform staining of ultrathin sections on grids.

A standard LKB (LKB-Produkter Ab. S-161, 25 Brommma 1, Sweden) grid storage box is converted into several grid staining boxes by sawing the body of the box into segments along rows of its grid storage cavities. The staining boxes can be cut out to any required size or shape. The polymethyacrylate storage box cover is discarded. Covers for the staining boxes are cut from thin sheet vinyl, which is more chemically resistant than polymethyacrylate. Corresponding 2 mm diameter holes are drilled through the vinyl covers and the bottoms of the grid storage cavities of the staining boxes to convert the storage cavities into staining chambers. For staining, the covers are tied to the boxes with sewing thread and the assembled units are put into vials. The separate staining chambers prevent intermingling of and mechanical damage to grids during the staining procedure. Ultrathin sections are more cleanly and uniformly stained in bulk by the use of these staining boxes than they are when stained individually by a standard method.

An ultrastructural study on the rôle of the keratohyalin granules in the keratinization process

Electron staining with phosphotungstic acid added to the alcohol used for the dehydration and combined with the staining of ultrathin sections with uranyl acetate or nitrate gives a high contrast-effect of different structures of Araldite-embedded skin. This investigation consists of an electron microscopic study of keratohyalin granules in normal epidermis of guinea pigs. The cytoplasm of stratum granulosum becomes progressively looser in texture as it approaches the skin surface. The tonofilament bundles in this layer show a low contrast, and seem to be split up and reduced in number. They are oriented around the keratohyalin granules. In the entire non-cornified epidermis are observed particles about 100–250 Å in diameter and arranged in groups. In stratum granulosum they are found around the keratohyalin granules. The latter are of different shapes and sizes and are scattered over the whole cytoplasm. They seem to grow through the incorporation of tonofilaments and the above-mentioned particles. Transitional cells (T-cells) between stratum granulosum and stratum corneum have been demonstrated in the dorsal and foot skin of guinea pigs. In dorsal skin they appear singly. Their cytoplasm is mainly filled with keratohyalin granules. Around these are seen groups of particles, apparently in close contact with the granules. The keratohyalin granules in granular and T-cells show a pattern. This is assumed to represent a precursor state in the development of the keratin pattern. The cell boundaries of T-cells show high opacity and a width of about 150 Å. The intercellular space between granular, T- and cornified cells contains a well-stained amorphous material in which vesicles are dispersed.