Mounting Medium Research Articles

Effect of heat stress during in vitro maturation on developmental competence of vitrified bovine oocytes.

Heat-shock protein 70 (HSP70) plays a crucial role as intracellular cytoprotectant and molecular chaperone. A phenomenon of heat stress (HS) leads to production of these proteins that could be beneficial to cells during cryopreservation, which is also a stressful process for the cell. This study aimed to evaluate the protective effect of exposure of bovine oocytes to moderate HS during in vitro maturation (IVM) prior vitrification. First, oocytes were subjected to HS (41.5°C for 1hr) at 0, 4, 8, 12 or 16 of IVM. Oocytes in vitro matured for 20hr served as control group. Presence of HSP70 was detected at 20hr by immunofluorescence. HSP70 expression was significantly higher when oocytes were subjected to HS at 8hr of IVM. Next, oocytes were distributed into four groups: Control: IVM oocytes; VIT: oocytes vitrified/warmed at 20hr of IVM; HS: oocytes subjected to HS at 8hr of IVM; HS-VIT: oocytes subjected to HS at 8hr of IVM and vitrified/warmed at 20hr of IVM. Oocytes were fertilized at 24hr of IVM, and cleavage and blastocyst yield were assessed. No significant differences were observed among treatments when cleavage rate was evaluated. However, fresh control and HS oocytes resulted in a significantly higher (p<.05) blastocyst rate when compared to VIT and HS-VIT groups, although no significant differences within fresh or vitrified groups were observed. In conclusion, HS did not have a negative impact on the oocyte competence but HS applied before vitrification, offered no benefits for embryo development.

Viability of fungi in boric acid glycerol methylene blue mounting medium

Viability of fungi in boric acid glycerol methylene blue mounting medium

Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations

A wide range of aspects concerning microscope slides, their preparation, long-time storage, curatorial measures in collections, deterioration, restoration, and study is summarized based on our own data and by analyzing more than 600 references from the 19th century until 2016, 15 patents, and about 100 Materials Safety Data Sheets. Information from systematic zoology, conservation sciences, chemistry, forensic sciences, pathology, paleopathology, applied sciences like food industry, and most recent advances in digital imaging are put together in order to obtain a better understanding of which and possibly why mounting media and coverslip seals deteriorate, how slides can be salvaged, which studies may be necessary to identify a range of ideal mounting media, and how microscope studies can benefit from improvements in developmental biology and related fields. We also elaborate on confusing usage of concepts like that of maceration and of clearing. The chemical ingredients of a range of mounting media and coverslip seals are identified as much as possible from published data, but this information suffers in so far as the composition of a medium is often proprietary of the manufacturer and may vary over time. Advantages, disadvantages, and signs of deterioration are documented extensively for these media both from references and from our own observations. It turns out that many media degrade within a few years, or decades at the latest, except Canada balsam with a documented life-time of 150 years, Euparal with a documented life-time of 50 years, and glycerol-paraffin mounts sealed with Glyceel, which represents almost the only non-deteriorating and easily reversible mount. Deterioration reveals itself as a yellowing in natural resins and as cracking, crystallization, shrinkage on drying or possibly on loss of a plasticizer, detachment of the coverslip, segregation of the ingredients in synthetic polymers, as well as continued maceration of a specimen to a degree that the specimen virtually disappears. Confusingly, decay does not always appear equally within a collection of slides mounted at the same time in the same medium. The reasons for the deteriorative processes have been discussed but are controversial especially for gum-chloral media. Comparing data from conservation sciences, chemical handbooks, and documented ingredients, we discuss here how far chemical and physical deterioration probably are inherent to many media and are caused by the chemical and physical properties of their components and by chemicals dragged along from previous preparation steps like fixation, chemical maceration, and physical clearing. Some recipes even contain a macerating agent, which proceeds with its destructive work. We provide permeability data for oxygen and water vapor of several polymers contained in mounting media and coverslip seals. Calculation of the penetration rate of moisture in one example reveals that water molecules reach a specimen within a few days up to about a month; this lays to rest extensive discussions about the permanent protection of a mounted specimen by a mounting medium and a coverslip seal. Based on the ever growing evidence of the unsuitable composition and application of many, and possibly almost all, mounting media, we strongly encourage changing the perspective on microscope slides from immediate usability and convenience of preparation towards durability and reversibility, concepts taken from conservation sciences. Such a change has already been suggested by Upton (1993) more than 20 years ago for gum-chloral media, but these media are still encouraged nowadays by scientists. Without a new perspective, taxonomic biology will certainly lose a large amount of its specimen basis for its research within the next few decades. Modern non-invasive techniques like Raman spectroscopy may help to identify mounting media and coverslip seals on a given slide as well as to understand ageing of the media. An outlook is given on potential future studies. In order to improve the situation of existing collections of microscope slides, we transfer concepts as per the Smithsonian Collections Standards and Profiling System, developed for insect collections more than 25 years ago, to collections of slides. We describe historical and current properties and usage of glass slides, coverslips, labels, and adhesives under conservational aspects. In addition, we summarize and argue from published and our own experimental information about restorative procedures, including re-hydration of dried-up specimens previously mounted in a fluid medium. Alternatives to microscope slides are considered. We also extract practical suggestions from the literature concerning microscope equipment, cleaning of optical surfaces, health risks of immersion oil, and recent improvements of temporary observation media especially in connection with new developments in digital software.

Abstract 3831: The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology

Abstract Introduction When a mouse or monoclonal antibody is desired for immunohistochemical detection on mouse tissues, the anti-mouse secondary antibody will also bind to endogenous mouse IgG in the tissue and may produce unwanted background staining. In the past, mouse-on-mouse (MM) detection utilized a biotinylated primary antibody or biotinylation kit. Tissues rich in endogenous biotin such as kidney or liver made it difficult to use, as streptavidin-horseradish peroxidase is required for detection. Attempts to completely negate endogenous biotin are ineffective as avidin-biotin blocking steps are unlikely to work acceptably in problematic tissues. Therefore, the design of a digoxigenin-labeled detection system is advantageous for MM detection as it would eliminate these issues. This technology has broad application as it allows the use of a variety of monoclonal antibodies reactive on mouse tissues. Materials and Methods Mouse tissues were formalin-fixed for 24 hours and embedded in paraffin (FFPE). Sections were cut and dried on slides before deparaffinizing and peroxidase blocking in the usual manner. Slides were then HIER in a citrate-based buffer prior to application of antibody. Mouse antibody concentrates were selected and mixed with a Digoxigenin Anti-Mouse Linker and incubated at room temperature (RT) for 1 hour. An Absorption Reagent is then added to the mixture, and further incubated for 30 minutes. The mixture is then diluted in antibody diluent to the desired concentration and applied to the mouse tissue for 60 minutes at RT, followed by a rabbit anti-digoxigenin antibody for 15 minutes. Detection of anti-digoxigenin is performed with an incubation in either a goat anti-rabbit HRP or AP Polymer for 30 minutes. Visualization is accomplished by using a DAB or Fast Red chromogen. For double stains, a sequential DS method using two monoclonal antibodies was employed using a heating step in retrieval solution after the first chromogenic reaction followed by a second monoclonal antibody and detection with either a rabbit-AP or HRP polymer. Visualization of the second antibody target is accomplished by using Fast Red or a blue chromogen. Slides were coverslipped with a permanent mounting medium and evaluated using brightfield microscopy. Results MM single stains with a variety of antibodies were easily achieved with specific, clean and biotin-free staining. Sequential DS techniques using monoclonal antibodies were also achieved and visualized with DAB, Fast Red or blue chromogens. Both manual and automated procedures were used. Conclusion The replacement of biotin-labeled MM technology with a digoxigenin MM technology is a superior method that not only eliminates endogenous biotin, but allows utilization of HRP and AP polymers for double stain technology. Citation Format: Joseph Vargas, David Tacha, Sara Figueroa. The development of an immunohistochemical digoxigenin-labeled mouse on mouse single and double stain methodology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3831. doi:10.1158/1538-7445.AM2017-3831

Influence of preservative and mounting media on the size and shape of monogenean sclerites.

Based on Cichlidogyrus sp. (Monogenea, Ancyrocephalidae) specimens from Hemichromis sp. hosts, we tested the influence of different methods to fix/preserve samples/specimens [frozen material, alcohol or formalin preserved, museum process for fish preservation (fixed in formalin and preserved in alcohol)] and different media used to mount the slides [tap water, glycerin ammonium picrate (GAP), Hoyer's one (HM)] on the size/shape of sclerotized parts of monogenean specimens. The results show that the use of HM significantly increases the size of haptoral sclerites [marginal hooks I, II, IV, V, and VI; dorsal bar length, width, distance between auricles and auricle length, ventral bar length and width], and changes their shape [angle opening between shaft and guard (outer and inner roots) in both ventral and dorsal anchors, ventral bar much wider, dorsal one less curved]. This influence seems to be reduced when specimens/samples are fixed in formalin. The systematics of Monogenea being based on the size and shape of their sclerotized parts, to prevent misidentifications or description of invalid new species, we recommend the use of GAP as mounting medium; Hoyer's one should be restricted to monogenean specimens fixed for a long time which are more shrunken.

ZPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain.

In studies of brain function, it is essential to understand the underlying neuro-architecture. Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used. We describe here a modified version of the passive clearing technique (PACT) ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ; Treweek et al., 2015) , which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) ( Frétaud et al., 2017 ; Mayrhofer et al., 2017 ; Xavier et al., 2017 ), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) ( Dambroise et al., 2017 ), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) ( Fini et al., 2017 ) . This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others ( Chung et al., 2013 ; Tomer et al., 2014 ; Yang et al., 2014 ), was adapted for use in aquatic species, including zebrafish in particular (zPACT). This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity. The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) ( Ke et al., 2013 ) , with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.

Differentiation of populations with different fluorescence intensities with a machine-learning based classifier

As proven fast and reproducible, cellular diagnostics based on machine learning has gained importance lately by its capability to predict different classes of cells based on cell and nuclear morphology, replacing specific markers. The present study aimed to verify if it is possible to differentiate objects with different fluorescence intensities using a machine learning based classifier. Samples were prepared with one drop of fluorescent mounting medium and bead solution (cytocal). Images were generated in .tif format using iCys image cytometer and saved for all relevant channels of fluorescence. Two softwares were used to analyze the samples, CellProfiler (CP) and CellProfiler Analyst (CPA). CP was used for segmentation of the samples and exported to a sQ-lite format database to run on CPA. CPA was used to classify the machine learning of six different peak classes of intensity and one class of artifacts. Also, using CPA, sensibility was calculated and a histogram of the intensity peaks was generated. CPA identified correctly nearly 100 out of 100 beads in each population, resulting in a total sensibility of 0.98. Artifacts resulted in a sensibility slightly lower than 1. It can be concluded that the database exportation between the two softwares does not lose fluorescent intensity quality. In addition, CPA is a useful instrument to verify morphologically quantitative data from CP. At last, CPA is completely able to identify correctly up to seven different intensity populations.

Primary cilia are present on human blood and bone marrow cells and mediate Hedgehog signaling.

Primary cilia are nonmotile, microtubule-based organelles that are present on the cellular membrane of all eukaryotic cells. Functional cilia are required for the response to developmental signaling pathways such as Hedgehog (Hh) and Wnt/β-catenin. Although the Hh pathway has been shown to be active in leukemia and other blood cancers, there have been no reports describing the presence of primary cilia in human blood or leukemia cells. In the present study, we show that nearly all human blood and bone marrow cells have primary cilia (97-99%). In contrast, primary cilia on AML cell lines (KG1, KG1a, and K562) were less frequent (10-36% of cells) and were often shorter and dysmorphic, with less well-defined basal bodies. Finally, we show that treatment of blood cells with the Hh pathway ligand Sonic Hedgehog (SHh) causes translocation of Smoothened (SMO) to the primary cilia and activation of Hh target genes, demonstrating that primary cilia in blood cells are functional and participate in Hh signaling. Loss of primary cilia on leukemia cells may have important implications for aberrant pathway activation and response to SMO inhibitors currently in clinical development.

Abstract 3144: Detection of HER2-HER3 heterodimers in patient circulating tumor cells using proximity ligation assay

Abstract BACKGROUND: The HER2 protein is a key oncogene in approximately 20% of breast cancers, and preferentially forms heterodimers with other members of the HER family (such as HER3) to activate its signaling cascade. Dual HER2 signaling blockade by using both trastuzumab and pertuzumab (a HER2 dimerization inhibiting monoclonal antibody) yields superior clinical benefit in HER2+ patients compared to trastuzumab monotherapy. A phase 1/2 clinical trial is ongoing to assess the safety and efficacy of the combination of gemcitabine with trastuzumab and pertuzumab in metastatic HER2+ pretreated patients. (NCT02139358) One of the correlative experiments is looking at the feasibility of detecting HER2-HER3 heterodimers using a proximity ligation assay on circulating tumor cells isolated from patients on treatment as a possible pharmcodynamic marker. METHODS: Whole blood was collected in two 10cc Cellsave tubes (Veridex, Cat#: 7900005) at baseline, week 4, 7, 10. Samples were drawn before treatment was administered, to avoid contamination of the samples with cytotoxic agents. The blood was subjected to red cell lysis with 1XRBC lysis buffer (Santa Cruz). The lysed cells were fixed with 4% paraformaldehyde followed by negative selection utilizing CD45+ magnetic beads (EasySep CD45 depletion kit, cat#18259). The CD45 depleted cell suspension was re-fixed with 4% PFA on coated glass slides following cytospin. These cells were then stained for PLA to test for HER2-HER3 using Sigma Aldrich DuoLink PLA kit + DAPI fluorescence mounting medium (DUO92013) with anti-HER2 (PA5, Thermo Scientific, diluted 1:50) and anti-HER3 (MA5-12675, Thermo Scientific diluted 1:50) as primary antibodies plus cytokeratin (CK) and CD45 antibodies. A set of HER2+ and HER2- PLA controls using SKBR3 were included in the analysis. Control samples using SKBR3 cells were used to optimize the method. RESULTS: Using the spiked SKBR3 HER2+ blood samples it was possible to isolate CK+ CD45- DAPI+ CTCs demonstrating HER2-HER3 heterodimers following optimization of the PLA method. Using this method in the first four patients we successfully collected CTCs in 7/11 samples processed. In those 7 CTC+ samples, 4 contained CTCs with positive HER2-HER3 PLA signals. CONCLUSIONS: We have demonstrated a feasible methodology for the detection of HER2-HER3 heterodimers on the surface of circulating tumor cells from HER2+ metastatic breast cancer patients. This can provide investigators with a non-invasive method to monitor the pharmacodynamics of pertuzumab therapy on tumor cells. The collection of these samples is ongoing and correlation with benefit from therapy will be analyzed in the phase 2 portion of the study. Citation Format: Hatem Soliman, Fatema Khambati. Detection of HER2-HER3 heterodimers in patient circulating tumor cells using proximity ligation assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3144.

Raman spectroscopy as a tool for the collection management of microscope slides

Throughout the history of preparation of biological samples for microscopy the choice of the mounting medium was sometimes dictated merely by availability of the used media. Thus, a plethora of resins and other organic polymers as well as complex mixtures are found to serve as mounting agents in microscope slide collections of museums of natural history, impeding the work for both curators and conservators. Dramatically, in some cases the used mounting media can already be observed to have undergone crystallization and other decomposition processes within few years of mounting demanding immediate action in restoring as well as an imminent precaution in conservation. Therefore, an unambiguous chemical identification of the used agent as well as its current aging stage is of great interest for the biologist community. The technical demands on the analytical approach to obtain this information can be straightforwardly identified. Any used technique has to be non-destructive, yield in molecular information allowing for a chemical identification of the used mounting agents and allow for a spatially well-defined interrogation in a thin sample slice, typically through a transparent cover slip. In this contribution we present a thorough study of the applicability of Raman spectroscopy for the described task. The obtained results clearly demonstrate the successful feasibility of the chosen method for a) a clear distinction between different media, b) the elucidation of the chemical composition of a multicomponent medium and c) an unambiguous identification of real unknown samples by a distinct assignment to a previously recorded spectral library. This library database was built up by recording pure mounting agents and will be provided to the general public. In combination with a Raman spectrometer, it can be an invaluable tool for future curation and conservation endeavors devoted to microscope slide collections at natural history museums.

Laser-scanning microscopy as applied to mouse early embryos: Cytometry and analysis of cell morphology

This paper updates our knowledge on quantitative laser scanning microscopy and summarizes the capabilities of this method as applied to cytometry and analysis of cell structure of the mouse early embryo and the oocyte. This method requires a stack of optical sections obtained as Z-series with subsequent 3D reconstruction. This approach was used for visualization of the 3D cell model, measurement of the cell volume and surface area, as well as a study of the cell interior via optical sections. To maintain the dimensional characteristics of embryos or oocytes the method of sample preparation involved the following consequent steps: rapid cryofixation, low-temperature dehydration, infiltration by optically transparent mounting media, and laser-scanning microscopy. This strategy enables volume measurement, even in the case of a single cell within the multicellular system of the mouse early embryo.

Cuticle and subsurface ornamentation of intact plant leaf epidermis under confocal and superresolution microscopy.

Plant cuticle micromorphology is an invaluable tool in modern ecology and paleoecology. It has expanded our knowledge of systematic relationships among diverse plant groups and can be used to identify fossil plants. Furthermore, fossil plant leaf micromorphology is used for reconstructing past environments, most notably for estimating atmospheric CO2 concentration. Here we outline a new protocol for imaging plant cuticle for archival and paleoecological applications. Traditionally, both modern reference and fossil samples undergo maceration with subsequent imaging via environmental SEM, widefield fluorescence, or light microscopy. In this paper, we demonstrate the capabilities of alternative preparation and imaging methods using confocal and superresolution microscopy with intact leaf samples. This method produces detailed three-dimensional images of surficial and subsurface structures of the intact leaf. Multiple layers are captured simultaneously, which previously required independent maceration and microtome steps. We compared clearing agents (chloral hydrate, KOH, and Visikol); mounting media (Eukitt and Hoyer's); fluorescent stains (periodic acid Schiff, propidium iodide); and confocal vs. superresolution microscopes. We conclude that Eukitt is the best medium for long-term preservation and imaging. Because of nontoxicity and ease of procurement, Visikol made for the best clearing agent. Staining improves contrast and under most circumstances PAS provided the clearest images. Supperresolution produced higher clarity images than traditional confocal, but the information gained was minimal. This new protocol provides the botanical and paleobotanical community an alternative to traditional techniques. Our proposed workflow has the net benefit of being more efficient than traditional methods, which only capture the surface of the plant epidermis. Microsc. Res. Tech. 81:129-140, 2018. © 2016 Wiley Periodicals, Inc.

Deficiency of IκB Kinase β in Myeloid Cells Reduces Severity of Experimental Autoimmune Encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), peripherally developed myelin-reactive T lymphocytes stimulate myeloid cells (ie, microglia and infiltrated macrophages) to trigger an inflammatory reaction in the central nervous system, resulting in demyelination and neurodegeneration. IκB kinase β (IKKβ) is a kinase that modulates transcription of inflammatory genes. To investigate the pathogenic role of IKKβ in MS, we developed strains in which IKKβ was conditionally ablated in myeloid cells and established active or passive EAE in these animals. Deficiency of IKKβ in myeloid cells ameliorated EAE symptoms and suppressed neuroinflammation, as shown by decreased infiltration of T lymphocytes and macrophages and reduced inflammatory gene transcription in the spinal cord at the peak or end stage of EAE. Myeloid deficiency of IKKβ also reduced the transcription of Rorc or Il17 genes in T lymphocytes isolated from lymph nodes, spleen, and spinal cord of EAE mice. Moreover, cultured splenocytes isolated from myeloid IKKβ-deficient EAE mice released less IL-17, interferon-γ, and granulocyte-macrophage colony-stimulating factor after treatment with myelin peptide than splenocytes from IKKβ wild-type EAE mice. Thus, deficiency of myeloid IKKβ attenuates the severity of EAE by inhibiting both the neuroinflammatory activity and the activation of encephalitogenic T lymphocytes. These results suggest IKKβ may be a potential target for MS patients, especially when neuroinflammation is the primary problem.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Evaluation of immunohistochemical staining for glucagon in human pancreatic tissue

Immunohistochemistry (IHC) and immunofluorescence (IF) staining techniques are important diagnostic tools of anatomic pathology in the clinical setting and widely used analytical tools in research laboratories. In diabetes research, they are routinely used for the assessment of beta- and alpha-cell mass, for assessment of endocrine cell distribution within the pancreas, for evaluation of islet composition and islet morphology. Here, we present the evaluation of IHC techniques for the detection of alpha-cells in human pancreatic tissue. We compared the Horse Radish Peroxidase (HRP)-based method utilizing DAB Peroxidase Substrate to the Alkaline Phosphatase (AP)-based method utilizing Vector Red substrate. We conclude that HRP–DAB staining is a robust and reliable method for detection of alpha-cells using either rabbit polyclonal or mouse monoclonal anti-glucagon antibodies. However, AP-Vector Red staining should be used with caution, because it is affected by the dehydration with ethanol and toluene preceding the mounting of slides with Permount mounting medium. When AP-Vector Red is a preferable method for alpha-cell labeling, slides should be mounted using aqueous mounting medium or, alternatively, they could be air-dried before permanent mounting

Mounting ground sections of teeth: Cyanoacrylate adhesive versus Canada balsam.

Introduction:Hard tissues can be studied by either decalcification or by preparing ground sections. Various mounting media have been tried and used for ground sections of teeth. However, there are very few studies on the use of cyanoacrylate adhesive as a mounting medium.Aims:The aim of our study was to evaluate the efficacy of cyanoacrylate adhesive (Fevikwik™) as a mounting medium for ground sections of teeth and to compare these ground sections with those mounted with Canada balsam.Materials and Methods:Ground sections were prepared from twenty extracted teeth. Each section was divided into two halves and mounted on one slide, one with cyanoacrylate adhesive (Fevikwik™) and the other with Canada balsam. Scoring for various features in the ground sections was done by two independent observers.Statistical Analysis Used:Statistical analysis using Student's t-test (unpaired) of average scores was performed for each feature observed.Results:No statistically significant difference was found between the two for most of the features. However, cyanoacrylate was found to be better than Canada balsam for observing striae of Retzius (P < 0.0205), enamel lamellae (P < 0.036), dentinal tubules (P < 0.0057), interglobular dentin (P < 0.0001), sclerotic dentin – transmitted light (P < 0.00001), sclerotic dentin – polarized light (P < 0.0002) and Sharpey's fibers (P < 0.0004).Conclusions:This initial study shows that cyanoacrylate is better than Canada balsam for observing certain features of ground sections of teeth. However, it remains to be seen whether it will be useful for studying undecalcified sections of carious teeth and for soft tissue sections.

A Novel PD-L1-targeting Antagonistic DNA Aptamer With Antitumor Effects.

The PD-1/PD-L1 axis is a major pathway involved in tumor immune evasion. Here, we report the novel PD-L1 antagonizing DNA aptamer (aptPD-L1) and demonstrate an integrated pipeline that expedites therapeutic aptamer development. Aptamer can exert antibody-mimic functions and is advantageous over antibody for its chemically synthetic nature, low immunogenicity, and efficient tissue penetration. Our results showed that aptPD-L1 blocked the binding between human PD-1 and PD-L1. Experiments using murine models showed that aptPD-L1 promoted in vitro lymphocyte proliferation and suppressed in vivo tumor growth without the induction of observable liver or renal toxicity. Analyses on the aptPD-L1-treated tumors further revealed elevated levels of infiltrating CD4+ and CD8+ T cells, intratumoral IL-2, TNF-α, interferon (IFN)-γ and the C-X-C motif chemokines, CXCL9 and CXCL10. The CD8+ T cells in the aptPD-L1-treated tumors had higher CXCR3 expression level compared to the random-sequence oligonucleotides-treated ones. Besides, the length and density of CD31+ intratumoral microvessels were significantly decreased in the aptPD-L1 treatment group. Collectively, our data suggested that aptPD-L1 helps T cell function restoration and modifies tumor microenvironment. These chemokines might orchestrate together to attract more T cells into the tumor tissues to form the positive amplification loop against tumor growth, indicating the translational potential of aptPD-L1 in cancer immunotherapy.

Hematoxylin Counterstain To Simplify Whole Slide Scanning Of Immunofluorescence Stains

Introduction/ Background Whole slide scanning of immunofluorescence (IF) stained slides is a well-recognized need, because fluorescence signal fading makes slides non-archivable. Technically scanning of IF has remained difficult, because tissue finding and autofocus operations are slow and prone to errors under epifluorescence illumination. Aims To simplify and to make IF scans more reliable, we started to look for counterstains that are compatible with IF and that can be scanned under brightfield illumination first. Methods Of the many alternative counterstains tested, light hematoxylin (H) proved the best counterstain for IF (with or without DAPI). Hematoxylin staining allowed the scanner (OIT Turboscanner) to perform rapid and accurate tissue finding and to define autofocus points, yet it did not increase background autofluorescence or decrease true fluorescence signal during subsequent scanning of IF under epifluorescence illumination. By using Cy2 (green) and Cy3 (orange) as secondary antibody fluorochromes (Jackson Immunotech), we were able to use standard xylene-based DPX mounting medium without observing significant fluorescence signal fading. Results As a result, IF scans of H counterstained slides could not be distinguished from those stained without hematoxylin counterstaining. As a bonus, the two scans (IF and H) could be viewed on the viewer screen side-by-side, or by gradually blending the two layers with each other. Both viewing modes were found useful in diagnostic dermatopathology when analyzing e.g. blistering skin lesions. A brightfield-IF scanning combination was also found useful in double immunostains, where cellular co-localization makes use of two precipitating (chromogenic) markers unreliable. The peroxidase IHC-hematoxylin-IF stain was exemplified with Ki-67 and pan-cytokeratin in breast cancer samples. The brightfield WSI of Ki-67 with H counterstain is an easily evaluable chromogenic DAB stain, but the image analysis software (ImmunoRatio2, embedded in the WSI viewer) utilizes the hidden pan-cytokeratin IF to create a cancer mask to exclude counting of non-epithelial stromal and lymphoid cells in the Ki-67 labeling index counting. Together these results demonstrate that a minor modification in the staining protocol (using hematoxylin instead of or in parallel to DAPI as counterstain) provides a significant help for whole slide scanning of immunofluorescence stains. Some scanners may need to be re-programmed to allow scanning of brightfield and epifluorescence automatically in sequence.

Ethosuximide Induces Hippocampal Neurogenesis and Reverses Cognitive Deficits in an Amyloid-β Toxin-induced Alzheimer Rat Model via the Phosphatidylinositol 3-Kinase (PI3K)/Akt/Wnt/β-Catenin Pathway

Neurogenesis involves generation of new neurons through finely tuned multistep processes, such as neural stem cell (NSC) proliferation, migration, differentiation, and integration into existing neuronal circuitry in the dentate gyrus of the hippocampus and subventricular zone. Adult hippocampal neurogenesis is involved in cognitive functions and altered in various neurodegenerative disorders, including Alzheimer disease (AD). Ethosuximide (ETH), an anticonvulsant drug is used for the treatment of epileptic seizures. However, the effects of ETH on adult hippocampal neurogenesis and the underlying cellular and molecular mechanism(s) are yet unexplored. Herein, we studied the effects of ETH on rat multipotent NSC proliferation and neuronal differentiation and adult hippocampal neurogenesis in an amyloid β (Aβ) toxin-induced rat model of AD-like phenotypes. ETH potently induced NSC proliferation and neuronal differentiation in the hippocampus-derived NSC in vitro. ETH enhanced NSC proliferation and neuronal differentiation and reduced Aβ toxin-mediated toxicity and neurodegeneration, leading to behavioral recovery in the rat AD model. ETH inhibited Aβ-mediated suppression of neurogenic and Akt/Wnt/β-catenin pathway gene expression in the hippocampus. ETH activated the PI3K·Akt and Wnt·β-catenin transduction pathways that are known to be involved in the regulation of neurogenesis. Inhibition of the PI3K·Akt and Wnt·β-catenin pathways effectively blocked the mitogenic and neurogenic effects of ETH. In silico molecular target prediction docking studies suggest that ETH interacts with Akt, Dkk-1, and GSK-3β. Our findings suggest that ETH stimulates NSC proliferation and differentiation in vitro and adult hippocampal neurogenesis via the PI3K·Akt and Wnt·β-catenin signaling.

Fatigue induced changes in conical implant–abutment connections

ObjectivesBased on the current lack of data and understanding of the wear behavior of dental two-piece implants, this study aims for evaluating the microgap formation and wear pattern of different implants in the course of cyclic loading. MethodsSeveral implant systems with different conical implant–abutment interfaces were purchased. The implants were first evaluated using synchrotron X-ray high-resolution radiography (SRX) and scanning electron microscopy (SEM). The implant–abutment assemblies were then subjected to cyclic loading at 98N and their microgap was evaluated after 100,000, 200,000 and 1 million cycles using SRX, synchrotron micro-tomography (μCT). Wear mechanisms of the implant–abutment connection (IAC) after 200,000 cycles and 1 million cycles were further characterized using SEM. ResultsAll implants exhibit a microgap between the implant and abutment prior to loading. The gap size increased with cyclic loading with its changes being significantly higher within the first 200,000 cycles. Wear was seen in all implants regardless of their interface design. The wear pattern comprised adhesive wear and fretting. Wear behavior changed when a different mounting medium was used (brass vs. polymer). SignificanceA micromotion of the abutment during cyclic loading can induce wear and wear particles in conical dental implant systems. This feature accompanied with the formation of a microgap at the IAC is highly relevant for the longevity of the implants.