Mounting Medium Research Articles

Chironomid Pupal Exuviae Technique in Ecological Research of Man-Made Water Bodies

Reservoirs serve functional purposes such as irrigation and power generation. However, concerns are raised due to the alterations of the connected riverine ecosystems. Chironomidae (Diptera), a diverse aquatic macroinvertebrate group, are vital to the functioning of ecosystems and serve as water quality indicators. Their holometabolous development includes the pupal stage after four larval stages. The chironomid pupal skin (exuvia) is used in environmental assessments, where the Chironomid Pupal Exuvial Technique (CPET) is a recognized standard. The CPET method is adaptable to different freshwater environments and here was applied in the study of 28 man-made lakes in the Pannonian Lowlands and Dinaric Western Balkan Ecoregion in Croatia to obtain information on chironomid diversity and analyze the potential influence of environmental factors on the chironomid community. The lake surface was skimmed with an exuvial hand net (mesh size of 300 µm) along the lake edge with a transect length of 10 m in the area of accumulated debris of organic and inorganic matter. Individual exuviae were mounted in a Berlese mounting medium and identified by morphological characteristics to the lowest taxonomic level. During the study, 5698 chironomid pupal skins were collected, and 141 taxa (including 97 species) belonging to five subfamilies were identified. The tribe Tanytarsini comprised 40% of the identified taxa, with Paratanytarsus spp. being the most abundant. In the Dinaric ecoregion, Paratanytarsus bituberculatus dominated, while Microchironomus tener and the genus Cricotopus were the dominant taxa in the Pannonian ecoregion. Community structure in the Pannonian ecoregion was influenced by total organic carbon (TOC) and orthophosphates (PO43−), indicating higher anthropogenic pressure compared to the Dinaric ecoregion, where water conductivity influenced Chironomidae assemblages. The research has provided valuable and useful information on the chironomid diversity in man-made and highly altered water bodies, as some of the most vulnerable aquatic habitats to anthropogenic influence. The CPET method could be a useful tool for the ecological studies and bioassessment of water quality in Croatia.

Innovative sample preparation using alcohol dehydration and high refractive index medium enables acquisition of two-channel super-resolution 3D STED image of an entire oocyte.

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and large biological structures have posed a challenge. To overcome this, the key step is to optimise sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique two-channel 3D STED SR image of an entire mouse oocyte. By optimising sample preparation, it is possible to overcome current limitations of SR methods and obtain high-resolution images of large biological structures, such as oocytes, in order to study fundamental biological processes. Lay Abstract: Super-resolution (SR) microscopy is a cutting-edge tool that allows scientists to view incredibly fine details in biological samples. However, it struggles with larger, thicker specimens, as they need to be optically clear and uniform for the best imaging results. In this study, we refined the sample preparation process to make it more suitable for SR microscopy. Our method includes carefully dehydrating biological samples with alcohol and then transferring them into a mounting medium that enhances optical clarity. This improved protocol enables high-resolution imaging of thick biological structures, which was previously challenging. By optimizing this preparation method, we hope to expand the use of SR microscopy for studying large biological samples, helping scientists better understand complex biological structures.

A novel m-xylylene-diamine/glucose based-supramolecular eutectogels with tissue clearing for three dimensional histological imaging

Hydrogel-based tissue clearing technologies have shown significant promise for deep-tissue imaging and subcellular-level optical 3D reconstruction of whole organs. This study proposes a novel approach utilizing a deep eutectic solvent (DES) formulated with glucose and m-xylylene-diamine (MXDA) to create a highly efficient tissue-clearing hydrogel system named the passive hydrogel clearing system (PHCS). PHCS achieved efficient tissue clearing through a single-step tissue gelation process. The resulting hydrogel-tissue complex exhibited thermoreversible properties, transitioning into a sol state upon heating and vice versa upon cooling. Notably, PHCS enabled media embedding, facilitating immunofluorescence histopathology. Additionally, the system demonstrated compatibility with various fluorescent probes, particularly lipophilic dyes. Our study successfully employed PHCS for the reconstruction of vascular structures within the intestine, enabling the generation of a 3D pathology model. These findings suggest that PHCS is a promising novel method for fabricating hydrogels for tissue clearing and holds great potential for application as a mounting medium for morphological imaging.

Tuning the excitation laser power in a stochastic optical reconstruction microscope for Alexa Fluor 647 dye in Vectashield mounting media.

Super-resolution imaging techniques have fundamentally changed our understanding of cellular architecture and dynamics by surpassing the diffraction limit and enabling the visualization of subcellular details. The popular super-resolution method known as stochastic optical reconstruction microscopy (STORM) relies on the exact localization of single fluorescent molecules. The significance of employing Vectashield as a mounting medium for the super-resolution imaging scheme called direct STORM has recently been explored. Alexa Fluor 647 (AF647), one of the most popular dyes, shows significant blinking in Vectashield. However, to observe prominent blinking of the fluorophore for the reconstruction of super-resolved images, the power of the excitation laser needs to be tuned. This work demonstrates the tuning of excitation power density in the sample plane for superior imaging performance using AF647 in Vectashield. Samples comprising MDA-MB-231 breast cancer cell line are used for the experiments. The actin filaments of the cell are stained with phalloidin-conjugated AF647 dye. For the experiment, we employ a low-cost openFrame-based STORM system equipped with a programmable Arduino-regulated laser source emitting at 638nm. An excitation power density of 0.60 kW/cm2 at 638nm in the sample plane is observed to maximize the signal-to-noise ratio, the number of switching events, and the number of photons detected per event during image acquisition, thereby leading to the best imaging performance in terms of resolution. The outcome of this work will promote further STORM-based super-resolved imaging applications in cell biology using Alexa Fluor 647 in Vectashield.

Anatomy of a fossil liana from the Upper Cretaceous of British Columbia, Canada

Summary Fossil woods are preserved as calcareous permineralizations in the Upper Cretaceous Northumberland Formation on Collishaw Point, Hornby Island, British Columbia, Canada. They are well preserved, often including peripheral tissues, and reveal the diversity and structure of vegetation in the region ca. 72 million years ago. We describe a new genus and species based on two specimens. Two specimens were cut using a rock saw to expose the fossil wood in transverse and longitudinal sections. Thin sections were prepared using the cellulose acetate peel technique. Peels were mounted on slides with Eukitt mounting medium. Anatomical description follows the IAWA guidelines. We compared the anatomy of the fossils with that of other plants using data available in the published literature and the InsideWood database. The combination of wide vessels and high vessel frequency given the narrow diameter of the stem, combined with the abrupt increase in vessel diameter near the pith indicates this plant had hydraulic properties typical of a climbing plant (liana). The anatomy of the rays, the arrangement of the vessels and their pitting, and the thickness of the bark are also consistent with the climbing habit, but not diagnostic of any extant family. This is the second species of liana to be described from the Hornby Island assemblage, providing new information about the composition of structurally complex mixed conifer–angiosperm forests in the Late Cretaceous of western North America.

Practical considerations for birefringence microscopy of myelin structure: Microscope design and tissue processing for effective imaging

Abstract Despite the interest in studying and quantifying the structural integrity of myelin in postmortem brain tissue, current methods for high-resolution imaging of myelin with optical microscopy are not sufficient. While imaging methods must have adequate resolution and sensitivity to detect microstructural alterations to myelin that are relevant in aging and neurodegenerative disease, an equally critical aspect is to minimize myelin damage that is induced during tissue processing steps. Birefringence microscopy (BRM) is a powerful technique that leverages the structural anisotropy of myelin to provide detailed, label-free images of myelin at any diffraction-limited optical resolution, while maintaining a simple and low-cost setup. Building on our previous work, we have developed a new BRM system and image processing pipeline that enable efficient, high-throughput imaging of myelin structure at multiple scales. Here, we utilize this system to systematically assess the damage to myelin that is induced by several common tissue processing steps in brain sections from the rhesus monkey. Images taken of the same myelinated axons, before and after each tissue processing step, provide direct evidence that mishandling of tissue during sample preparation can cause significant structural alterations to myelin. First, we report on key advancements to our BRM system, imaging procedure, and image processing pipeline, which provide significant increases to the speed and efficiency of BRM. These include integrating fast piezoelectric rotational stages, minimizing the number of images required (to three images) for determining birefringence parameter maps, and implementing an analytical solution for directly determining birefringence parameter maps. Second, using this BRM system, we demonstrate that effective myelin imaging requires (1) the avoidance of prolonged drying or dehydration of tissue, (2) the selection of the optimal mounting medium (85% glycerol), (3) the avoidance of tissue permeabilization with detergents (i.e., Triton X-100 and Saponin), and (4) the selection of a suitable tissue-section thickness (15, 30 and 60 µm) based on the region of interest. In addition to serving as a guide for new users interested in imaging myelin, these basic experiments in sample preparation highlight that BRM is very sensitive to changes in the underlying lipid structure of myelin and suggest that optimized BRM can enable new studies of myelin breakdown in disease. In this work, we show that BRM is a leading method for detailed imaging and characterization of myelin, and we provide direct evidence that the structure of myelin is highly sensitive to damage during inadequate preparation of brain tissue for imaging, which has previously not been properly characterized for birefringence imaging of myelin. For the most effective, high-resolution imaging of myelin structure, tissue processing should be kept to a minimum, with sections prevented from dehydration and mounted in 85% glycerol. With proper preservation of myelin structure, BRM provides exquisitely detailed images that facilitate the assessment of myelin pathology associated with injury or disease.

#2281 SEM for the diagnosis of glomerular diseases (amyloidosis, minimal change, thin membrane, IgA, membranoproliferative and diabetic nephropathy)

Abstract Background and Aims Treating renal diseases often requires ultrastructural details to establish or confirm diagnosis. Ultrastructural information that is not evidenced by optical microscopy includes, e.g., the podocyte foot processes effacement, thinning or alteration of basement membrane, presence of fibrils, Fabri's intracellular inclusions and subepithelial vs. subendothelial of deposits. Unfortunately, transmission electron microscopy (TEM), required to achieve the required resolution, has high costs, and is time-consuming. Here, we report our experience with scanning electron microscopy (SEM) on paraffin sections from standard pathology sections. Method We have retrospectively reanalyzed paraffin sections from renal biopsies of 50 patients with a diagnosis of IgAN, minimal change, diabetes, and amyloidosis. The paraffin sections were available and processed according to standard procedures (fixation, paraffin embedding), with a thickness of 3-4 µm. Sections were deparaffinized, stained with PASM (periodic acid Shiff + methenamine), mounted in aqueous mounting medium and imaged with 40x and 60x objectives. Sections were then unmounted, washed to remove glycerol, dehydrated in absolute alcohol, and air-dried. Finally, they were gold coated (“sputtering”) and analyzed in a scanning electron microscope (Zeiss). Results For the interpretation of SEM images, correlative analysis has been used, imaging precisely the same region of the section using standard optical microscopy and SEM. SEM can quickly identify the typical morphology of podocyte foot processes (Fig. 1A) and the podocyte effacement in minimal change disease (Fig. 1B). SEM can also easily allow the measurement of the thickness of the basement membrane of glomerular capillary walls (Fig. 1C-E) and its thinning in case of thin membrane disease (Fig. 1D) or its increased thickness in the case of diabetes (Fig. 1E). Amyloid fibrils (Fig. 1F) can also be identified in the capillary walls, confirming the diagnosis of amyloidosis Congo-red positive (Fig. 1G). The high resolution of the technique allows us to identify with high reliability the small ruptures of the capillary walls in IgA and membranoproliferative nephritis, with extravasation of erythrocytes and fibrin cloths (Fig. 1H). Mitochondria are well-demarcated, particularly in proximal tubule cells, and often, mitochondrial cristae are visible in samples with adequate fixation (Fig. 1I). Interstitial edema and fibrosis are evident in SEM microscopy (Fig. 1L). At very high magnification, in histological preparations with exceptional fixation, the filtration pores of endothelial cells could be observed (partially visible in Fig. 1E), as well as details such as the brush border of the tubular epithelial cells. The interpretation of the images is not always straightforward as it also requires knowledge of the artifacts induced by the fixation, processing, and inclusion of paraffin, such as, for example, the intracytoplasmic cellular structures containing lipids of the intratubular “foam cells” appear empty. For a correct interpretation of the SEM images, however, the recognition of the nuclei is essential because the chromatin is condensed into clods, especially around the periphery, and inside, some areas appear empty. Conclusion Scanning electron microscopy (SEM) is a powerful tool for the study of nephrological diseases, as it allows you to examine microscopic details at the nanometric scale (not detectable in optical microscopy), providing valuable information for diagnosis, understanding, and management of the disease, the possibility of carrying out Correlative Microscopy. Given the relatively simple and fast preparation of the samples to be examined, the low costs of execution, and the possibility of archiving the preparation in the histotheque for a possible revision of the case, this leads us to think that this method could be considered a valid alternative to transmission electron microscopy (TEM).

Detecting SARS-CoV-2 in bats of New Mexico using immunohistochemistry on formalin- fixed, paraffn-embedded tissues

Objectives: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) and its zoonotic origin have raised critical questions regarding the potential role of local bat populations as reservoirs or intermediates for the transmission of this or related coronaviruses to humans. The goal of this project is to investigate the presence and diversity of coronaviruses in bat species native to the Southwest USA. Broadly, this study has two primary aims: (1) to detect antigens of coronaviruses in tissues of local bat populations via histological methods and (2) to perform a molecular screen of coronaviruses in these bats. Methods: Individuals from five bat species were evaluated: Artibeus jamaicensis, Myotis velifer, Antrozous pallidus, Tadarida brasiliensis, and Eptesicus fuscus. All species were local, wild-caught bats, with the exception of Artibeus jamaicensis, which is from a colony established at New Mexico State University (NMSU). Tissue was sourced from bats collected by TJO at NMSU. Bat tissue was fixed with formalin and samples subsequently taken of lung, kidney, nasal epithelium, and uterus. Tissue was further processed and embedded in paraffn wax. The embedded tissue was sectioned via a rotary microtome at 4 μm thickness and mounted onto slides. Slides were stained with general coronavirus anti-nucleocapsid primary antibody and a hematoxylin counterstain to visualize cellular structures. Slides were cover-slipped with DPX mounting medium and observed via light microscopy. Results: The immunohistochemistry protocol was performed on tissue from each of the five bat species investigated and on human placenta samples known to be positive for SARS-CoV-2. The latter was used as positive controls. Putative positive staining was seen in both human placenta and bat tissues. Interestingly, coronavirus was detected in Artibeus jamaicensis samples, an unexpected finding given its status as a laboratory colony. Verification of this putative finding is ongoing. Further work on our samples will determine the genetic identity of the coronaviruses detected and contribute to developing a molecular screen for more effcient detection of coronavirus antigens in local bat tissue. This work will serve as a springboard for further understanding coronavirus ecology and evolution in bats of the US southwest, and it can be used to further inform both local surveillance efforts and public policy surrounding the interaction between humans and animals in the natural environment and in controlled settings. Conclusions: Putative detection of coronavirus antigens was found in tissues from bat samples from the Southwestern USA. Implications for this work include broadening our understanding of the diversity and abundance of coronaviruses in the US. Burrell College; US National Science Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 6886: Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma

Abstract Background: The ability to image tissue microenvironments (TME) at high-plex over a whole slide without requiring multiple staining steps allows for unprecedented ability to study tissue architecture and molecular mechanisms of immune and disease processes. Here we investigate a sample of normal lung section adjacent to non-small cell lung carcinoma using whole slide, single-step stain and imaging at single-cell resolution. Methods: These images profiled a whole slide tissue section of normal lung section adjacent to non-small cell lung carcinoma, stained with a 17-plex immuno-oncology biomarker panel. In this profile, we designed a high-plex panel of 17 biomarkers where the tissue autofluorescence was imaged and isolated as an additional fluorescence channel. All markers were stained in a single staining procedure and imaged in a single scan. Whole slide spatial staining and imaging was conducted on the Orion spatial biology platform. The full protocol is quick and simple, using standard histology tools: •Mount sections on glass slides•De-paraffinize and perform antigen retrieval•Quench autofluorescence•Stain slides with a panel of ArgoFluor™ conjugated antibodies•Coverslip with ArgoFluor Mounting Medium and cure overnight•Image whole slides at 20X magnification using the Orion instrument•Process to ome.TIFF and analyze. Results: Data revealed intact normal lung alveolar architecture, with scattered immune cell infiltrates. Multiplexed imaging revealed epithelial cells, which make up alveoli, and endothelial vasculature as well as immune cells infiltrates in the alveolar space. Various immune cell populations were observed to be present (T cells, B cells, macrophages) and immune cell activation could be distinguished using PCNA and/or Ki-67 markers. Various T cell subsets could also be identified among the infiltrate using markers for CD4, CD8 and T-regulatory marker FOXP3. Through the use of multiplex staining, immune cell activity could also be characterized with respect to bronchiolar, artery, and capillary tissue architecture. Conclusions: High-quality subcellular imaging of TME can lead to greater spatial investigation and more in-depth understanding of immune function in normal and diseased tissue. Here, multiplexed imaging identified distinct immune cell collections among cancer-adjacent normal lung tissue, providing validation of instrumentation and methods to obtain benchmarks for spatial quantitation of immune cells in control regions adjacent to cancer tissue. Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Orion™: 17-plex single-step stain and imaging of normal lung section adjacent to non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6886.

Abstract 5502: Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types

Abstract Background: Tissue consists of heterogenous cell types, each with diverse functions and functional states, where spatial organization can impact patient health status. Resolving this complexity at the subcellular level has historically been challenged by image resolution, the number of targets that can be simultaneously assessed, and throughput. Such barriers can be broken using high plex and whole slide biomarker imaging data, in one single cycle thus also mitigating issues that arise with multiple cyclic rounds. Methods: A range of whole slide healthy and diseased tissue types were stained with 15 to 18-plex validated biomarker panels. The tissues’ inherent autofluorescence was isolated into discrete fluorescence channels for enhanced information about each tissue’s morphology. Whole slide spatial staining and imaging was conducted on the Orion™ spatial biology platform, and H&E staining was performed after immunofluorescence (IF) imaging on the same section and imaged by brightfield microscopy. The full protocol is fairly quick and simple, using standard histology tools: - Mount sections on glass slides- De-paraffinize and perform antigen retrieval- Quench autofluorescence- Stain slides with a panel of ArgoFluor™ conjugated antibodies- Coverslip with ArgoFluor Mounting Medium and cure overnight- Image whole slides at 20X magnification using the Orion instrument- Process to ome.TIFF and analyze- De-coverslip in aqueous solution- Perform H&E staining and scanning on same section. Results: Imaging data within each tissue type presented here reveals important biological insights which can then be used for quantitative analysis. The little to no tissue degradation due to the one round staining and imaging process also enables H&E capabilities after IF, further enhancing biomarker quantitation. The combination of speed and resolution proved effective for comprehensive phenotypic profiling and characterization of tissue architecture, tumor heterogeneity, and the immune response. Conclusions: There is a need to show tissue biology in a high quality, high resolution, subcellular fashion as it provides a more in-depth understanding of each biological sample and thus can lead to improved spatial research of various human diseases. The single-stain and scan method described here improves the acquisition of subcellular spatial context and greatly reduces/removes errors in multiplex image data compared to cyclic methods. A single stain and image cycle across a 12-18 plex sample provides accurate downstream biomarker quantitation, offering better prediction of patient outcomes. Citation Format: Selena Larkin, Tad George, Eric Kaldjian. Multiplex data utilizing a single-step staining and imaging workflow for the investigation of multiple sample types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5502.

Abstract 7635: Orion™: 14-plex clinical sample imaging of liver cancer modalities using one-step staining and imaging

Abstract Background To better understand the complexities of the tumor microenvironment (TME), high-resolution imaging at a sub-cellular resolution and high-plex is needed. These serve as a basis for biomarker quantification and more downstream, may provide the ability to predict patient outcome. Here, we investigate three whole slide liver sections: fetal liver, hepatocellular carcinoma (HCC), and colorectal cancer (CRC) liver metastasis. Methods 5-micron sections were cut and mounted on Superfrost Plus slides prior to de-paraffinization and antigen retrieval using the BioGenex EZ-Retriever system. Autofluorescence was quenched using UV and white light and blocked with Image-iT™ FX Signal Enhancer. Whole slides were stained with the 14-plex antibody panel, coverslip mounted with ArgoFluor™ Mounting Medium and cured overnight. Whole slides were imaged at 20X using the Orion™ spatial biology platform. Coverslips were removed in aqueous solution prior to H&E staining and scanning. Results Sample specific differences were observed, likely stemming from the different tissue sources. Data revealed a distinction between hepatocytes highlighted in the autofluorescence channel, sinusoids filled with proliferating progenitor cell and macrophages, and epithelial cells lining the border of the fetal liver forming the ductal plate surrounding the portal vein. Whereas in HCC, imaging revealed autofluorescent hepatocytes obliterating the sinusoids with some hepatocytes dividing, an extensive capillary network, and red bloods cells also highlighted in the auto fluorescent channel. T cells and macrophages showed clustering within the capillary networks and as expected, hepatocytes expressed high levels of Albumin. In the CRC liver metastasis sample, hepatocytes were shown to be surrounded by T cells, neoplastic glands (PanCK), and CD163+FOLR2+ macrophages. Furthermore, the majority of the metastatic epithelial cancer cells were observed to be proliferating. Conclusions We were able to observe precise differences in the tissue architecture of liver samples from three divergent sources. Specifically, the ability to identify distinct cell subtypes and their spatial co-localization may provide mechanistic insights into the spatial immune landscape and its role in disease progression and treatment outcome. This exemplifies the need for dissecting the spatial heterogeneity of the TME using a technology that offers a high-resolution subcellular snapshot in time; exhibiting the potential for Orion to bridge the gap from bench-to-bedside. Citation Format: Selena Larkin, Jennifer Currenti, Rhea Pai, Soumi Chatterjee, Archita Mishra, Jacob George, Brady Gardner, Ankur Sharma. Orion™: 14-plex clinical sample imaging of liver cancer modalities using one-step staining and imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7635.

Using disposable food packaging materials as printing, embedding, and sectioning media in the plant anatomy lab.

During the COVID-19 pandemic lockdown, all laboratory work was suspended, and we were obliged to work from home, causing delays in our research. As the disruption to supply chains made it difficult to obtain our regular lab supplies, we were obliged to search for substitutes. We became familiar with a plastic material known as biaxially oriented polypropylene (BOPP) that is widely used in the food industry for wrapping or storing fruits, vegetables, and meat. BOPP is easily dissolved in organic solvents such as xylenes, acetone, or thinner, but these reagents are very toxic, flammable, and can cause nausea in some users. After testing several alternatives, we found a polyurethane remover that proved to be an effective and relatively harmless BOPP solvent. By dissolving thin slices of BOPP in a polyurethane solvent, we obtained a clean fluid that we used to obtain leaf surface prints that could be mounted on microscope slides with a coverslip. This fluid produced excellent bark and wood sections and can be used to obtain wood or charcoal surface prints. Our attempts to use it as a mounting medium were unsuccessful. BOPP dissolved in a polyurethane remover is a handy, versatile resource for plant microtechniques. In addition to its economic advantages, it is useful in terms of reducing plastic pollution.

Neuroplasticity in honey bee brains: An enhanced micro-computed tomography protocol for precise mushroom body volume measurement

BackgroundIn insect brains, mushroom bodies are associated with memory and learning behavior. It has been demonstrated that the volume of the mushroom bodies in the brain of a worker honey bee changes during the adult stage. Changes in mushroom body volume imply high neuroplasticity in the brains and may be related to the age polyethism of honey bees. A suitable volume measurement method is needed to understand the correlation between behavioral changes and mushroom body volume changes in honey bees. New methodWe developed a new protocol for insect micro-computed tomography by modifying a previously reported method. Permount™ mounting medium was used as the embedding medium for micro-computed tomography scanning. ResultsThis protocol can generate images with high contrast inside the brain and reduce the marked shape changes during specimen processing. From the resulting high-contrast images, we used freeware to generate a three-dimensional model and calculate the volumes of the mushroom bodies in honey bees. The measured volumes of the mushroom bodies were larger than the values reported in most previous studies. There was no significant difference between the left and right mushroom body volumes, but the volumes of honey bee mushroom bodies significantly increased with age. Comparison with existing methodsPrevious protocols for micro-computed tomography using dried samples would cause brain shrinkage; protocols using ethanol-preserved or resin-embedded samples generated images with lower contrast. ConclusionsThe embedding protocol for micro-computed tomography is suitable for calculating volume of the mushroom bodies in honey bee brains.

The use of ethyl cellulose film lifts to collect particle traces from exposed adhesive on the edges of duct tape

Environmentally acquired particles (EAP), trapped along the edges of duct tape in the exposed adhesive, are a possible source of information regarding prior exposures of the tape and a possible means to associate duct tape rolls to segments of duct tape that are collected as traces during the investigation of criminal activity. The recovery and separation of EAP is complicated by (1) the need to separate the particles from the adhesive, and (2) the presence of adhesive filler and pigment particles that are part of adhesive formulations. Approaches such as cutting the tape edge, followed by solvent extraction, or swabbing of the duct tape edges using solvents, result in thousands of adhesive filler/pigment particles, overwhelming much smaller numbers of EAP. A recovery method has been developed employing a 10% solution of ethylcellulose (EC) in ethanol. Duct tape segments are pressed onto a glass plate and a bead of the viscous EC solution is pipetted along the edge. After drying, the EC forms a robust film that is peeled away from the tape edge, lifting the EAP along with only very small amounts of adhesive. The film lift (containing EC, duct tape adhesive and EAP) can be examined directly by steromicroscopy and particles of interest can be removed by softening the film using locally applied ethanol. Particles can be examined in situ by transmitted light microscopy using glycerol as a mounting medium to match the EC film. Batch recovery of particles from the film can be achieved by dissolution of the film lift in xylene and washing to remove adhesive residues. Xylene is then removed by washing with ethanol, leaving an ethanol suspension of EAP along with the duct tape filler/pigment particles. Larger particles can be separated by sieving and smaller particles can be recovered by filtration. This method was found to efficiently collect EAP found trapped along the edges of duct tape in the exposed adhesive, as well as for the preparation of these particles for microscopical and instrumental analysis. Limitations, not fully evaluated, include the loss or alteration of some particle types by dissolution, agitation and filtration steps. Further development of this method is expected to provide a means to recover trace particles from the edges of other types of tape and from related adhesive products such as stamps and labels.

Qualitative Evaluation of Modified Polyvinyl Alcohol Gel Used as A Preservative Medium Compared to Traditional Modified Kaiserling Iii Solution in Pathology Museum

Pathology and anatomy museums preserve body tissues and organs traditionally in modified Kaiserling III solution and display in transparent glass jars, which serve as valuable teaching materials for students. Other techniques tried in preserving specimens are embedding in molten wax, embedding in epoxy resin, and plastination techniques. Modified Carbopol-940 gel has been tried recently as a preservative mounting medium in pathology museum. However, the use of modified Polyvinyl alcohol (PVA) gel as a preservative medium is not mentioned in the literature. Gel preservative medium can prevent leakage, toxic vapors, and irritation of eyes and skin. Aims: Qualitative evaluation of modified Polyvinyl alcohol gel as a preservative medium for specimens in pathology museums compared to modified Kaiserling III solution over one year. Objectives: 1) To qualitatively evaluate physical integrity of mounted tissue specimens stored in modified Kaiserling III solution and modified Polyvinyl alcohol gel for one year. 2) To qualitatively evaluate physical, chemical and microbiological parameters of modified Kaiserling III solution and modified Polyvinyl alcohol gel for one year. Materials and methods: In this qualitative cross-sectional study, six different organs, two sets each, were preserved in jars containing modified Kaiserling III solution and modified Polyvinyl alcohol gel. The media were evaluated for stability by physical tests, chemical test, and microbiological test, before and after the study. The specimens were assessed for physical disintegration by observing for tissue shredding, and evaluated for histopathological appearances, before and after the study. Results showed mild variability in stability of the preservative media but good tissue integrity of the specimens over a period of one year. 

Silica and barium: Comparison of microscopic appearance and characterization of effects of silica on cytomorphology.

Silica from plastic red top sample collection tubes and barium cause recognized artifacts in slide preparations for microscopic examination. The objectives of this study were to evaluate and directly compare the microscopic appearance of silica and barium particles and various slide preparation techniques (e.g., use of coverslips, oil immersion, and different stains). A secondary objective of this study was to evaluate the effects of silica particles on cellular morphology after mechanical trauma with cytocentrifugation. Fluid samples (deionized water, pleural effusion, peritoneal effusion, cerebrospinal fluid, and urine) were collected and evaluated in silica- and non-silica-containing tubes. Barium was added to silica and non-silica samples. Direct and cytocentrifuge preparations were compared to evaluate the effect of silica particles on cellular morphology. Preparations were stained with Wright-Giemsa, rhodizonic acid disodium salt, Alizarin Red, Grocott's methenamine silver, and Prussian blue. Silica and barium particles were identifiable via light microscopy with and without polarized light, although silica particles diminished with immersion oil. Barium particles retained their structure and diminished less under oil. Cytoseal mounting medium for coverslip placement resulted in diminished refractility of silica and some barium particles. Silica particles with mechanical interaction during cytocentrifugation resulted in disrupted cellular morphology with many lysed cells. Silica and barium particles were negative for all special stains tested. Silica from plastic red top tubes adversely affects cell morphology in cytocentrifuge preparations, potentially affecting manual differential cell counts and compromising diagnostic interpretation. Samples intended for microscopic evaluation should not be collected in silica-containing tubes.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.Graphical

Automation of slide coverslipping for large tissue sections.

Coverslipping is the process of placing a cover glass or coverslip over a glass slide mounted with a stained tissue specimen without forming air bubbles, which can negatively impact the microscopic examination. While manual coverslipping is still widely used, automated systems have made the process easier and more consistent. Commercially available automated cover-slippers are limited to handling only slides that are 1" x 3", suitable for processing smaller tissue specimens. However, for larger tissue specimens sectioned from organs like the brain, liver, etc., slides can reach sizes up to 6" x 8", exceeding the capabilities of these systems. We present SLIDE PROTEKT, a fully automated large format coverslipping system designed to efficiently coverslip large format slides. This system has multiple zones for slide and coverslip transportation, dispensing of mounting medium, and precise placement of the coverslip without air bubbles. The ability of the system to output quality coverslipped slides was validated by processing 50 large-format brain tissue slides. The results were found to be comparable to manual coverslipping. The system achieved a coverslip placement accuracy of 80% with a mean positional offset that was within a tolerance of ±3 millimeters. Additionally, 75% of the slides had no air bubbles, while the remaining slides had air bubbles that were less than 120 micrometers in size. These results demonstrate the potential impact of SLIDE PROTEKT in the field of histology.

P-203 The Effect of c-Abl Tyrosine Kinase Inhibition in Cell Fate Specification and Morphogenesis During Preimplantation Mouse Embryo

Abstract Study question What are the mechanisms governing the regulation of c-Abl tyrosine kinase in preimplantation embryo development? Summary answer Inhibition of c-Abl impairs the fate determination of TE and ICM cells during mouse preimplantation embryo development, preventing cell specilization after compaction. What is known already The successful completion of polarization, compaction and lineage specification stages is required for the embryo to reach the blastocyst stage from the zygote stage during preimplantation embryonic development. c-Abl is a tyrosine kinase localized in cell nucleus and cytoplasm and also capable of nuclear-cytoplasmic shuttling. c-Abl is activated when DNA damage occurs and plays a role in the DNA repairment mechanism. The small-molecule inhibitor imatinib is classified as type II inhibitors which targets the inactive conformation of the kinase domain and specifically inhibit c-Abl. Study design, size, duration C57BL6 female mice at 6-8-wk old were superovulated with 7,5 IU PMSG and after 48 hr 7,5 IU hCG, then mated. 20 hours after hCG enjection, zygotes were collected and cultured for 96h at 5% CO2, 5% O2 at 37oC. We designed 5 experimental groups: control, DMSO, 1 µM, 5 µM and 10 µM (in KSOM+AA) imatinib group. To determine how altering the imatinib might affect embryonic growth and development, morphokinetic parameters were evaluated. Participants/materials, setting, methods Immunofluorescence staining for embryos was performed after 4% paraformaldehyde fixation. Then embryos incubated c-Abl, YAP, TEAD4, PARD6, E-cadherin and CDX2. DAPI mounting medium used for nucleus staining. Imaging was performed under a confocal microscope. Fluorescent intensity analysis was performed with ImageJ. We detected c-Abl, Yap, Tead, Cdx2, Oct4 and Nanog mRNA expression levels using the qRT-PCR. For the statistical analysis we used GraphPad Prism (2-Way ANOVA and and One-way ANOVA (used Geisser-Greenhouse correction)). Main results and the role of chance Treatment of zygotes with imatinib resulted in embryonic developmental arrest (control, 81%; 1 µM imatinib, 58.5%; 5 µM imatinib, 54%, 10 µM imatinib 0%) and led to a significant decrease in the rate of blastocyst formation. We detected that the expression level of c-Abl was decreased in the imatinib groups compared to the control group. We determined that YAP and TEAD, which should be localized in the nuclei of TE cells, lost their nuclear expression patterns and were expressed in the cytoplasm and their expression levels decreased. We detected the PARD6 pattern, which was evident at the cell borders in the control group, distributed throughout the cytoplasm in the imatinib groups. While cytoplasmic and decreased localization of CDX2, which should be nuclear in TE cells, was determined in imatinib groups, we also defined that E-cadherin distribution was impaired at cell borders compared to the control group. c-Abl mRNA level was decreased in the 1 and 5 µm imatinib groups, Yap mRNA level was decreased in the 5 µm imatinib group. Cdx2 and Nanog mRNA levels were decreased in the 1 and 5 µm imatinib group (*p<0.1, **p <0.01, ***p <0.001, ****p <0.0001). Limitations, reasons for caution All animals used in this study were obtained from Yeditepe University Faculty of Medicine Experimental Research Center (YUDETAM) and all the experimental procedures have been approved by Yeditepe University Ethical Committee. Wider implications of the findings Imatinib significantly inhibited the development of eight-cell embryos to the blastocyst stage compared to controls a concentration-dependent manner, furthermore, c-Abl inhibition affected linegae specification by changing the localization of YAP/TEAD, markers of lineage differentiation. Therefore, c-Abl can regulate cell specification and cell fate determination during mouse preimplantation embryonic development. Trial registration number YAP-AP-SAB-21019

Efficacy of cyanoacrylate adhesive as a mounting medium for soft tissue sections as an alternative to Kirkpatrick and Lendrum's DPX

Introduction: Mounting is the final stage in histologically preparing a slide and creates a permanent the bond between the coverslip and the slide, preventing breakage, air drying artefacts, and stain fading. Materials & methods: In this study, fifty slides were prepared, twenty-five of which were mounted using DPX and twenty five using cyanoacrylate adhesive. The slides were assessed for clarity of nuclear and cytoplasmic details, adhesion between coverslip and slide, and the presence of air bubbles by two blinded observers. Results: The results showed that adhesion between coverslip and slide was better when mounted with cyanoacrylate adhesive, but better clarity of nuclear and cytoplasmic details and minimal air bubble entrapment was seen when tissue sections were mounted using DPX. Conclusion: Therefore, the use of cyanoacrylate adhesive cannot be an alternative to DPX for mounting soft tissue sections.