Spurr's Resin Research Articles

Simplified Sample Embedding and Polishing Methods for Preparing Hydrophilic, Fragile, or Solvent-Susceptible Materials for Thin Sections for Microscopic Analyses.

In the preparation of thin sections for microscopy, embedding and polishing processes in particular can change the composition and morphologies of samples. Soils and ashes are very fragile and solvent-susceptible, and appropriate sample preparation procedures have not been well-established. To improve the existing preparation methods and make them easier and faster, we embedded freeze-dried blocks, polished, and then examined these thin-section samples using polarization microscopy, laser microscopy, and field emission scanning electron microscopy with energy-dispersive X-ray spectrometry, and electron backscattered diffraction (EBSD). Appropriate thin-section samples can be prepared by: (1) rinsing with acetone and then embedding with Spurr resin along with repeated evacuation and ventilation, rather than conventional dehydration/replacement; (2) polishing using silicon carbide paper and diamond slurries, and then wiping with a cloth and a synthetic oil; and (3) slightly rinsing with 100% ethanol to remove the oil. The preparation method minimized contamination and pores, and showed flat surfaces and sometimes EBSD patterns. Freeze-drying has been claimed to cause the development of cracks due to ice crystal formation upon freezing, however, our method not only overcomes such problems for microscopic observation but saves substantial time, taking only 2 days in total to process a specimen, and requiring less than 1 g of resin and ~1 g of sample.

Anatomical structure of the leaves of Crassula cordata (Crassulaceae)

Crassulaceae DC. is a family of plants belonging to dicotyledons with a succulent leaves. They occur mostly in warm regions, especially many species distributed in Africa. Grow usually in sandy or mountainous areas and among the rocks, in habitats where water is scarce. The conditions which they live in cause that their leaves are modified to a greater or lesser extent. In a succulent leaf the chlorenchymatic tissue is not differentiated into the palisade and spongy layers. It forms clearly distinguishable water tissue with the large volumes of vacuoles. In leaf, additional amounts of water are stored as reserves during drought. The cells are tightly packed, the intercellular spaces are small. Such structure is also connected with the CAM photosynthesis. In Crassulaceae collecting of carbon dioxide takes place at night, and then used during photosynthesis during the day. It is adaptation for increased efficiency of water too. Moreover, in the large vacuoles, besides water, malate is accumulated – an intermediate compound in CAM. The epidermal tissue is also active in adaptation, often the outer call wall is thickened, covered a thick layer of cuticle, sometimes with a wax deposit. The aim of this study was to investigate the anatomy of the leaf of Crassula cordata Thunb. using the light microscopy. Mature leaves of plants were cut into small pieces and fixed in 2.5% glutaraldehyde and 2.5% formaldehyde in 0.1 M cacodylate buffer. Than tissue was post fixed in 2% osmium tetroxide, dehydrated in a graded acetone series and embedded in Spurr resin. Sections of 0.5-1.0 μm were cut using glass knives on a microtome and observed under light microscopy. Crassula cordata is a typical leaf succulent plant. It has a characteristic morphology and small size (Fig. 1). This also indicates the anatomical structure of the leaf, observed on the cross-sections (Fig. 2). Single-layered epidermis is composed of thick-walled, elongated cells. It is covered with a thick layer of cuticle. In the abaxial surface stomata was observed. Parenchyma is relatively uniform. The same cells perform the functions of water storage and photosynthesis. Mesophyll is not differentiated into palisade and spongy layer. Cells are tightly packed with small intercellular spaces. In mesophyll cells can be seen large vacuoles and plastids near the cell wall. These characteristics make good use of the volume for water storage. On the adaxial surface of leaf numerous hydathodes are present. Epithem, tissue which forms hydathodes, is composed of thick-walled, tightly packed, small cells with visible nucleus. Mesophyll cells surrounding vascular bundle and hydathodes contain vacuoles filled tannin substances.

First Report of Barley yellow striate mosaic virus on Wheat in China.

In the spring of 2014, a survey of viral diseases on wheat (Triticum aestivum L.) was carried out in Hebei Province, China. The samples with virus-like symptoms of dwarfing and flag leaf yellowing were collected in Zhaoxian, Quyang, Anxin, and Luannan. To reproduce the viral symptoms and confirm whether the unknown virus was transmitted by insect vectors, the nymphs of aviruliferous planthopper (Laodelphax striatellus Fallen, Homoptera: Delphacidae) were transferred onto diseased wheat from the field for a 3-day acquisition access period and a 10-day incubation on fresh wheat seedlings, and then were exposed to 2- to 3-leaf stage wheat seedlings of wheat variety Shixin828 for a 3-day inoculation access period. The infected wheat plants developed mosaic symptoms on the young leaves at 7 days post inoculation (dpi), and followed with severe symptoms including stunting, chlorotic spots, and striation along the veins of leaves at around 14 dpi. The infection symptoms were same as in the field but distinct from wheat infected with Rice black streaked dwarf virus (RBSDV) or Northern cereal mosaic virus (NCMV). For further confirmation, total RNA was extracted from the symptomatic wheat leaves, and NCMV specific primers, NCMV-PF/NCMV-PR (5'-ATGGATAAGAAAGCAAGTGGA-3'/5'-TTAAAAGTCGGCATACGGGTC-3') and RBSDV specific primers, S10-F/S10-R (5'-TTACCCAACATCACGCAACT-3'/5'-GAGCAGGAACTTCACGACAAC-3') were used for amplification of sequences of phosphoprotein and coat protein genes, respectively. Neither RBSDV nor NCMV were present in the symptomatic tissue according to the RT-PCR assay (4). Tissues derived from symptomatic wheat leaves were fixed and embedded in Spurr's resin and used for ultra-thin sectioning and transmission electron microscopy observations, revealing large amounts of Rhabdovirus-like particles in the cytoplasm. The identified particles were about 315 to 353 × 46 to 57 nm, similar in size to Barley yellow striate mosaic virus (BYSMV), a member of the genus Cytorhabdovirus reported from Italy (2). The specific primer pair (5'-ACTAAGGGGGTACTCCGACC-3' and 5'-CTGATCTGCTTTGAGGGGCA-3') was designed based on the reported polymerase (L) gene sequence of BYSMV isolate Zanjan-1 (GenBank Accession No. FJ665628) (1), and used for the BYSMV detection by RT-PCR. A single bright band of the expected size (~500 bp) was obtained from total RNA extracted from the plants exhibiting symptoms in the greenhouse. No such band was amplified from asymptomatic plants, while 15 out of 23 field samples also produced the same 500-bp products in RT-PCR. PCR products from three virus-positive field samples were sequenced directly and the sequences were submitted to GenBank (KM052176, KM052177, and KM052178). BLAST search showed that the sequences shared 96 to 97% nucleotide identity with the polymerase L gene sequence of BYSMV isolate Zanjan-1, whereas only 73 to 75% identity with NCMV (AB030277 and GU985153) (1,3,5). To our knowledge, this is the first report of BYSMV occurrence on wheat in China.

Ultrastructure of plant leaf cuticles in relation to sample preparation as observed by transmission electron microscopy.

The leaf cuticular ultrastructure of some plant species has been examined by transmission electron microscopy (TEM) in only few studies. Attending to the different cuticle layers and inner structure, plant cuticles have been grouped into six general morphological types. With the aim of critically examining the effect of cuticle isolation and preparation for TEM analysis on cuticular ultrastructure, adaxial leaf cuticles of blue-gum eucalypt, grey poplar, and European pear were assessed, following a membrane science approach. The embedding and staining protocols affected the ultrastructure of the cuticles analysed. The solubility parameter, surface tension, and contact angles with water of pure Spurr's and LR-White resins were within a similar range. Differences were however estimated for resin : solvent mixtures, since Spurr's resin is combined with acetone and LR-White resin is mixed with ethanol. Given the composite hydrophilic and lipophilic nature of plant cuticles, the particular TEM tissue embedding and staining procedures employed may affect sample ultrastructure and the interpretation of the results in physicochemical and biological terms. It is concluded that tissue preparation procedures may be optimised to facilitate the observation of the micro- and nanostructure of cuticular layers and components with different degrees of polarity and hydrophobicity.

Rice Lateral Root Development and Its Impact Factors

The adventitious roots and primary roots of Nipponbare and Yangdao 6 were used to measure lateral root number and the growth increment by Image J software with scanning photograph. Morphological and structural changes of lateral root primodia were observed by applying Spurr resin embedding and semi-thin sectioning, and light microscopy. Impacts of the lateral root emergence on the parent root structure was observed by fluorescence microscopy and scanning electron microscopy. Treatments with plant hormones, metal ions, illumination, and cutting off shoots or root tips were set up to analyse their influence on lateral root development. Results showed that rice lateral root initiation occurred from the pericycle. The endodermis also participated in the lateral root primodia formation and played protective as well as assistant roles in the process. The outward growth of lateral root primordia was realized by cell divisions of apical meristem and cell elongation at the base area. The cortex and epidermis structures of parent roots were also altered during the process of lateral root emergence. Exogenous IAA promoted the occurrence of lateral roots, but its high concentrations inhibited lateral root emergence. The number of lateral roots and growth of primary roots were significantly decreased when shoots were cut off. The apical dominance of root was lost when root tips were cut off, so that the growth of lateral roots and secondary lateral roots was accelerated. Unilateral illumination induced the increase of lateral roots in the light side. Abscisic acid at low concentrations promoted the formation of lateral roots. The formation of lateral roots and growth of primary root were promoted by Ca2+, while mostly inhibited by EDTA. The growth tendency of both adventitious roots and primary roots responsive to the treatments was basically consistent.

Effect of Both Local and Systemically Administered Dexamethasone on Long-Term Hearing and Tissue Response in a Guinea Pig Model of Cochlear Implantation

Dexamethasone administered prior to cochlear implantation has been shown to reduce the loss of residual hearing in experimental settings. However, its effect on the tissue response around the implant has not been extensively studied. In this study dexamethasone sodium phosphate was administered to guinea pigs via local delivery to the round window (2% dexamethasone for 120 min prior to surgery, ‘local 2/120', or 20% dexamethasone for 30 min prior to surgery) or intravenously (2 mg/kg dexamethasone for 60 min) prior to implantation. Auditory brainstem responses (ABR) were monitored for 3 months, after which the cochleae were embedded in Spurr's resin and sectioned. The extent of the tissue response and the survival of the neurosensory structures were analysed. Both local 2/120 and systemically delivered dexamethasone improved ABR thresholds when compared with control animals. Systemic dexamethasone also reduced the tissue response around the electrode. This suggests that whilst both locally and systemically administered dexamethasone can protect residual hearing after cochlear implantation, their effects upon the tissue response to implantation may differ.

Fine Structure of Bacterial Adhesion to the Epithelial Cell Membranes of the Filiform Papillae of Tongue and Palatine Mucosa of Rodents: A Morphometric, TEM, and HRSEM Study

The palatine mucosa and filiform papillae of the dorsal tongue mucosae of rodents were examined using transmission electron microscopy (TEM) and high resolution scanning electron microscopy (HRSEM). In the HRSEM method, the samples were fixed in 2% osmium tetroxide, dehydrated in alcohol, critical point-dried, and coated with gold-palladium. In addition, the HRSEM technique was used for morphometric analysis (length, width, and length/width ratio of cocci and bacilli). For the TEM method, the tissues were fixed in modified Karnovsky solution (2.5% glutaraldehyde, 2% formalin in 0.1M sodium phosphate buffer, pH 7.4) and embedded in Spurr resin. The results demonstrated that there are thick polygonal keratinized epithelial cells where groups of bacteria are revealed in three-dimensional images on the surface of filiform papillae in these animals. The bacterial membranes are randomly attached to the microplicae surface of epithelial cells. Morphometrics showed higher values of length and width of cocci in newborn (0 day) as compared to newborn (7 days) and adults animals, the bacilli showed no differences in these measurements. At high magnification, the TEM images revealed the presence of glycocalyx microfilaments that constitute a fine adhesion area between bacterial membranes and the membranes of epithelial microplicae cells. In conclusion, the present data revealed the fine fibrillar structures of bacteria that facilitate adhesion to the epithelial cell membranes of the oral cavity and morphometric changes in newborn (0 day) rats as compared with other periods.

Morphometric evaluation of the fetal rat liver after maternal dexamethasone treatment: effect on the maturation of erythroid and megakaryocytic cells

During pregnancy, glucocorticoids are frequently used to accelerate fetal lung maturation in preterm delivery. However, prenatal administration of glucocorticoids has been shown to affect organs such as fetal liver, an important hematopoietic organ during fetal development. The aim of this study was to document the qualitative and quantitative changes in erythroid and megakaryocytic cell populations found in fetal livers as well as the hematology profile in neonates after maternal glucocorticoid treatment in rats. Pregnant female Wistar rats were treated with dexamethasone 21-phosphate from days 13 to 16 of gestation. On the 17th day of pregnancy, the fetuses were collected and their livers processed for light and transmission electron microscopy. Glycol methacrylate-embedded sections were stained with PAS to determine the erythroblast and megakaryocytic cell frequencies. Fetal liver pieces embedded in Spurr resin were analyzed by transmission electron microscopy for morphologic changes. A standard hematology profile was evaluated in neonatal rats. In the fetuses from treated dams, the total cell number of erythroid cells in livers was significantly reduced compared to control fetuses (P < .001), but erythroblasts did not present ultrastructural abnormalities. The degree of maturation in the megakaryocyte series tended to be increased. In neonates, there were elevated numbers of nucleated RBCs (P = .002), along with a higher HCT and HGB (P = .02). In addition, the platelet concentration was also significantly increased (P < .007). These results suggest that maternal dexamethasone treatment has quantitative effects on erythroid and megakaryocytic cells in fetal liver and the neonatal hematology profile in rats.

Myocilin Mediates Myelination in the Peripheral Nervous System through ErbB2/3 Signaling

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

Immunolocalization of Smad-4 in developing molar roots of alendronate-treated rats

IntroductionDuring root formation, Smad-4 plays a key role during the epithelial–mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate. MethodsNewborn Wistar rats were daily injected 2.5mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope. ResultsIn ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation. ConclusionThe immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation.

Long Bone Development in the Japanese Quail (Coturnix coturnix japonica) Embryos

The current study was undertaken to describe the main histological development stages of long bones (tibia and femur) from Japanese quail (Coturnix coturnix japonica) embryos. Whole Limbs or just tibia and femur of fifty Japanese quail embryos of different ages were fixed and embedded in paraffin or Spurr's resin. Paraffin and semi-thin, respectively, were undertaken and examined with light microscopy. Limb bud was established at day 5 of incubation. Mesenchymal cells differentiated into chondrocytes forming a cartilage template in the position of the future tibia and femur at day 6 of incubation. At day 7 of incubation, the cartilage template enlarged and had the shape and position of the future tibia and femur. At day 8, central chondrocytes underwent hypertrophy and were surrounded by a periosteal bone collar. Cellular and vascular invasion from the bone collar into the central zone of the cartilage template, cartilage resorption and formation of marrow tunnel and finally peripheral calcification was seen. Vascular cartilage canals penetrating the epiphysis were observed at day 9 and the canals gradually increased in thickness and number toward the hatching day. Articular epiphyseal growth cartilage with resting, proliferative and hypertrophic zones was clearly established by day 10 of incubation. After 17 days of incubation, the zonation of the articular epiphyseal cartilage were much clear, many cartilage canals were present within the epiphyses. In epiphyses of tibia but not femur, foci of chondrocytes hypertrophy were noticed close to the cartilage canals. The current study timed the main histological sequences of development of tibia and femur of embryonic quail.

Immunohistochemistry and ultrastructural characteristics of nerve endings in the oral mucosa of rat

The sensory nerve endings of the rat tongue, cheek and palate were studied using immunohistochemical staining and transmission electron microscopy analysis. The specimens were fixed in modified Karnovsky solution and embedded in Spurr resin. Substance P, calcitonin gene-related peptide (CGRP)- and protein gene product 9.5 (PGP b9.5)-containing nerve fibers in the rat tongue, cheek and palate were examined by electronic microscopical analysis and immunohistochemical localization. These fibers run very close to the basal lamina of the epithelium and extend into the filliform and fungiform papillae. Numerous plexiform fibers immunoreactive for substance P, CGRP and PGP 9.5 were found in the connective tissue of mucosa. Electron microscopic observations showed clearly immunostained nerve fibers, which are located very close to the basal lamina of epithelial cells. Some electron-dense granules may be observed in the axoplasms of both substance P and CGRP immunoreactive fibers. Several lamellar corpuscles into the subepithelial connective tissue papillae, Merkel corpuscles and numerous thin unmyelinated and myelinated axons were observed. The terminal axons revealed numerous mitochondria, neurofilaments, microtubules and clear vesicles in the base of axoplasmic protrusions. The lamellar cells showed caveolae and interlamelar spaces filled by amorphous substance. Between the lamellar cells and axoplasmic membrane, and in the adjacent lamellae region, desmosome-type junctions were observed. The quantitative and morphometric analysis showed nerve endings with an average area of 4.83 ± 3.4 μm(2) and 19.4 internal mitochondria in this site and the organized corpuscles with an average area of 79.24 ± 27.24 μm(2) and 24.23 internal mitochondria in this place. All the structures observed are involved in the transmission of pain and mechanoreceptors stimulus of these oral mucosae.

Quantitative study on the hygroscopic expansion of spurr resin to obtain a high-resolution atlas of the mouse brain

Ultra-thin section-based microscopic imaging is considered one of the most realistic techniques for determining fine architectures of a brain-wide neural network. In this kind of method, the sample is usually embedded in resin and then immersed in water for sectioning and imaging. The effect of resin hygroscopic expansion on data accuracy and integrity is important as it may lead to inconsistent image qualities or degeneration of sectioning properties. But few studies have been conducted on this issue. Here, we have used surface profile measurements combined with sectioning and imaging by micro-optical sectioning tomography (MOST) to quantitatively study the sectioned surface expansion of spurr resin blocks as a result of water immersion for a short time period. The expansion effect on MOST imaging is also presented. The results revealed significant differences in the surface expansion of pure resin blocks with different immersion time durations (P < 0.001). During an eight-minute immersion, the surface expansion of the experimental specimens exhibited an approximately linear increase with immersion duration, while MOST images suffered a correlated decrease in brightness. Expansion was restricted to the submicron level with immersion duration of four minutes or less, and the mean and standard deviation of the expansion measurements both reached a maximum at eight minutes. When the immersion duration exceeded eight minutes, the expansion value decreased, which was most likely related to the degeneration of mechanical properties of the resin material on the block surface. This study indicates that it is necessary to select a specific sectioning mode according to the hygroscopic expansion properties of resin materials for maintaining the accuracy and integrity of whole brain atlas data.

Maternal corticosterone regulates nutrient allocation to fetal growth in mice

Stresses during pregnancy that increase maternal glucocorticoids reduce birth weight in several species. However, the role of natural glucocorticoids in the mother in fetal acquisition of nutrients for growth remains unknown. This study aimed to determine whether fetal growth was reduced as a consequence of altered amino acid supply when mice were given corticosterone in their drinking water for 5 day periods in mid to late pregnancy (day, D, 11-16 or D14-19). Compared to controls drinking tap water, fetal weight was always reduced by corticosterone. At D16, corticosterone had no effect on materno-fetal transfer of [(14)C]methylaminoisobutyric acid (MeAIB), although placental MeAIB accumulation and expression of the Slc38a1 and Slc38a2 transporters were increased. However, at D19, 3 days after treatment ended, materno-fetal transfer of MeAIB was increased by 37% (P < 0.04). During treatment at D19, placental accumulation and materno-fetal transfer of MeAIB were reduced by 40% (P < 0.01), although expression of Slc38a1 was again elevated. Permanent reductions in placental vascularity occurred during the earlier but not the later period of treatment. Placental Hsd11b2 expression, which regulates feto-placental glucocorticoid bioavailability, was also affected by treatment at D19 only. Maternal corticosterone concentrations inversely correlated with materno-fetal MeAIB clearance and fetal weight at D19 but not D16. On D19, weight gain of the maternal carcass was normal during corticosterone treatment but reduced in those mice treated from D11 to D16, in which corticosterone levels were lowest. Maternal corticosterone is, therefore, a physiological regulator of the amino acid supply for fetal growth via actions on placental phenotype.

Dendritic Cells Are Central Coordinators of the Host Immune Response to Staphylococcus aureus Bloodstream Infection

Dendritic cells (DCs) play an important role in integration of the immune responses induced by pathogens. The purpose of this study was to determine the importance of DCs in host defense against Staphylococcus aureus bacteremia. Using a murine infection model, we demonstrated that DCs are rapidly recruited into infected tissue after intravenous inoculation with S. aureus. The recruited DCs were fully functional and in a more advanced stage of maturation than those isolated from uninfected mice. Depletion of DCs in CD11c-DTR transgenic mice resulted in substantial worsening of infection, as indicated by increased bacterial loads in kidneys and lungs, accelerated mortality, and more severe pathology. Furthermore, DC depletion completely abolished IL-12 production in response to infection. The beneficial effect afforded by DCs during S. aureus infection was not mediated by their contribution to direct bacterial killing, nor by increased neutrophil recruitment. Instead, neutrophil influx (along with expression of CXC chemokines) was significantly enhanced in infected tissue after depletion of DCs. We also found that the bactericidal capacity of the recruited neutrophils was significantly impaired in DC-depleted mice. More importantly, the detrimental effect of DC depletion was practically reversed by treatment with exogenous recombinant mouse IL-12. Our results demonstrated that DCs, probably through their production of IL-12, play an important role in coordinating the inflammatory response during S. aureus infection.

Development of thallus axes in Usnea longissima (Parmeliaceae, Ascomycota), a fruticose lichen showing diffuse growth

While cell wall thickening in plants is generally associated with tissue maturation, fungal tissues in at least two lichens continue to grow extensively while accumulating massively thickened cell walls. We examined Usnea longissima to determine how diffuse growth shapes morphological and anatomical development of thallus axes and how the highly thickened cell walls of the central cord behave in diffuse growth. Fresh material was examined with light and epifluorescence microscopy and conventional and low-temperature SEM. Fixed material was embedded in Spurr's resin, microtome-sectioned, and examined with TEM and light microscopy. Main axes consisted essentially of bare medullary cord tissue; their characteristic morphology developed by destruction of the overlying cortex and consequent stimulation of lateral branch formation. Fungal cells of the cord tissue continually deposited wall layers of electron-transparent substances and layered, electron-dense materials that include UV-epifluorescent components. Discontinuities were evident in the outermost layers; new branch cells grew through wall materials accumulated by older neighboring cells. Sustained diffuse growth of cord tissue in U. longissima underlies the structural transformation of a corticated thallus branch into a long axis. In the cord tissue, diffuse growth may be responsible for the increasingly disrupted appearance of the older, electron-dense cell wall layers, while new wall materials are laid down adjacent to the protoplast. Cell and tissue development appeared comparable to that observed previously in Ramalina menziesii, although accumulation of wall material was somewhat less extensive and with a greater proportion of electron-dense/UV-epifluorescent components.

Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr's resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurr's resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done.

Effect of fixation and embedding methods on collagen fibrils diameter and spacing

AbstractPurpose The corneal tissue are usually fixed in fixatives and embedded in resin for ultrastructural studies. Here we study the effect of these fixatives and resins on the collagen fibril (CF) diameter and the spacing between the fibrils.Methods Four normal human post‐mortem corneal buttons of 24, 25, 54 and 75 year old individuals were used. A part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C under UV light for 24hrs. A second part of each cornea was fixed in glutaraldehyde (2.5%) + osmium tetroxide. The third part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer for ultrastructural studies. The tissues were embedded in spurr resin at 70°C for 12 hrs. Ultra‐thin sections were stained with uranyle acetate and lead citrate.Results In the tissue, fixed paraformaldehyde (4%) and embedded in LR White at 4°C under UV light for 24hrs, the CF diameter was 24± 0.1nm and spacing between CF was 39±0.1nm. In the tissue fixed in glutaraldehyde (2.5%) + osmium tetroxide, and embedded in spurr resin, the diameter was 28.37 ± 5.84nm and spacing between CF was 45 ±4.57nm. In the tissue, fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurr resin, the diameter was 33.75 ± 3.65nm and spacing between CF was 48.06 ±4.35nm.Conclusion Our study shows that there is a variation in the collagen fibril diameter and spacing depending on the method of fixation and embedding resins. This needs to be considered when comparative studies employing differ

Fine structure of myotendinous junction between the anterior belly of the digastric muscle and intermediate tendon in adults rats

This study analyzed the ultrastructural characteristics of the myotendinous junction (MTJ) between anterior belly of digastrics muscle and the intermediate tendon in adult rats. Six male Wistar rats were used and were anesthetized with an overdose of urethane and sacrificed by intracardiac perfusion with modified Karnovsky solution, postfixed in 1% osmium tetroxide, dehydrated in increasing series of alcohols and embedded in Spurr resin for transmission electron microscopic analysis. Ultrastructural analysis showed conical shape of the fiber extremity in MTJ region, highlighting the presence of numerous mitochondria arranged in groups in the subsarcolemmal and intermyofibrillary regions. Atypical MTJ characteristics were seen interspersed with bundles of collagen fibers. Classic characteristics such as finger-like processes by means of sarcoplasmic projections were observed among interdigitations. Terminals and periphericals bundles of myofibrils showed close relationship with the adjacent muscle fiber's endomysium through lateral junctions. In the distal portion, it was observed that the communication region of microtendons forming the intermediate tendon of digastric muscle, and it can highlight the columns disposition of tenocytes. In conclusion, the MTJ ultrastructure between the anterior belly of digastric muscle and intermediate tendon of adult rats showed classical morphologic descriptions and presented an atypical region revealed by the subspecialization between the myofibrils bundles and collagen fibers in the MTJ region.

Routing Misfolded Proteins through the Multivesicular Body (MVB) Pathway Protects against Proteotoxicity

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.