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Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. https://www.selleckchem.com/products/VX-770.html To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species' unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.Annual tree-ring patterns are a source of ecological and environmental information including the history of fires in forested areas. Tree-ring based fire histories include three fundamental phases field collection, laboratory methods (preparation and dating), and data analysis. Here we provide step-by-step instructions and issues to consider, including the process for selecting the study area, sampling sites, plus how and which fire-scarred trees to sample. In addition, we describe fire-scar sample preparation and dating which are done in the laboratory. Finally, we describe basic analysis and relevant results, including examples from studies that have reconstructed fire history patterns. These studies allow us to understand the historical fire frequency, changes in those frequencies related to anthropogenic factors, and analyzes of how climate influences fire occurrence over time. The description of these methods and techniques should provide a greater understanding of fire history studies that will benefit researchers, educators, technicians, and students interested in this field.
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