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Transmission electron microscopy significantly contributed to unveil the course of virus entry, replication, morphogenesis, and egress. For these studies, the most widely used approach is imaging ultrathin sections of virus-infected cells embedded in a plastic resin that is transparent to electrons. Before infiltration in a resin, cells must be processed to stabilize their components under the observation conditions in an electron microscope, such as high vacuum and irradiation with electrons. For conventional sample preparation, chemical fixation and dehydration are followed by infiltration in the resin and polymerization to produce a hard block that can be sectioned with an ultramicrotome. Another method that provides a superior preservation of cell components is high-pressure freezing (HPF) followed by freeze substitution (FS) before resin infiltration and polymerization. This chapter describes both procedures with cells infected with Bunyamwera virus (BUNV), a well characterized member of the Bunyavirales, and compares the morphological details of different viral structures imaged in the two types of samples. Advantages, disadvantages, and applications of conventional processing and HPF/FS are also presented and discussed., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In vivo models provide advantages to study the progression of disease and to identify potential biomarkers to detect and monitor infections. For the human fungal pathogen Cryptococcus neoformans, murine intranasal models aim to recapitulate natural infection from inhalation of desiccated fungal cells from the environment and permit monitoring of disease over time. In this chapter, we describe the establishment of a murine model for cryptococcosis and the subsequent collection of organs, tissues, and fluids for sampling. These samples may support novel diagnostic strategies and opportunities to monitor dissemination of the fungal cells throughout the host and propose new treatment options to combat disease., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter describes methodological details for preparing specimens of Cryptococcus neoformans (although it can be applied to any species of the genus) and their subsequent analysis by scanning and transmission electron microscopy. Adaptations to conventional protocols for better preservation of the sample, as well as to avoid artifacts, are presented. The protocols may be used to examine both the surface ultrastructure and the interior of this pathogenic fungus in detail., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Availability of relevant biological samples supports both basic science research and patient-centered clinical studies. Establishing a biorepository faces challenges at multiple levels. These tasks include defining mission definition and scope; selection of subjects and sample types; recruitment strategies; timing of collection in the patient's journey; sample logistics and processing; determining what clinical data to collect; ensuring sample integrity on transport, processing, and storage; defining governance structures and oversight responsibilities; clarifying sample provenance and ownership; establishing procedures for sample and data access; selecting testing to be performed routinely versus upon request, and management of results; data security; funding sources; and regulatory compliance. Establishing and maintaining a biorepository therefore requires careful planning, diligent and sustained execution, technical and financial resources, stakeholder support, and flexible and resilient management to respond to changing environments and needs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Immune monitoring of circulating immune cells in the blood provides insight into a patient's own immune response over the course of a treatment or disease progression. Information such as whether immune cells are functional or non-functional and what specific proteins they express or secrete can be essential to understand if (and how) a treatment is working or a disease is progressing. To do so, it requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples and minimizing future research costs by the use of banked samples. Many factors, including blood sample types, time of collection, containers used, preservatives and other additives, transport means, and length of transit time, all affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for genomic, immunological, and biochemical analyses. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the results. Here, we focus on the collection and processing of blood suitable for plasma and peripheral blood mononuclear cell (PBMC) banking, including collection, processing, and storage of samples, based on our experience., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The plasma and serum proteome has enormous potential as a tool for understanding the health of a number of physiological systems. Despite this potential, the use of plasma and serum proteomics clinically and for research is limited, and there are no strict guidelines on how samples should be collected and prepared for proteomic analysis. Given the sensitivity of proteomic analysis, there are a number of pre-analytical variables that should be considered and determined prior to undertaking proteomics-based methodologies.In this chapter, we provide an example of a blood processing protocol and highlight major considerations for pre-analytical variables involving the collection, processing, and handling of blood samples for plasma and serum proteomics. We provide comprehensive notes on aspects of the protocol that must be considered before commencing sample collections for a proteomic study as well as a thorough checklist to be used when designing new proteomic studies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

FTA ® cards enable efficient, long-term storage of blood and buccal cells/saliva samples for future forensic DNA analysis; these are typically collected as known reference samples, as opposed to evidentiary, crime scene samples. Upon contact with the FTA ® card, cells are lysed and the DNA is immobilized. Different FTA ® cards are available and have been specially formulated based on sample type: bloodstains are added to the traditional FTA ® Card, while colorless sources (e.g., buccal cells/saliva) are added to the FTA ® Indicating Card. The main difference between these cards is the presence of a pink dye embedded in the indicating cards that becomes white when exposed to colorless fluids, like saliva; this aids in location confirmation of the stain for future sampling. Although DNA can be eluted/extracted from FTA ® punches using various methods or, alternatively, direct STR amplification from unpurified punches can be performed, the protocol herein describes a simple purification method for bloodstained punches from FTA ® Cards as well as buccal/saliva-stained punches from FTA ® Indicating Cards. Following this purification, STR amplification can be performed via the "punch-in" method., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Regular monitoring of various biomarkers and molecular panels in plasma can significantly help to prevent disease onset and improve its management and final outcomes. Many groups can benefit from monitoring programs focusing on the prevention of cardiovascular diseases, evaluation of environmental exposure impacts, or the prevention/management of cancer. Improvement in therapeutic options in part due to targeted therapeutic agents and monoclonal antibody therapies has led to a significant sized population that can be described as "cancer survivors." These patients, although in remission from their original disease, are at significant risk for the recurring disease and must be monitored for adverse events. Monitoring is, however, not an easy task; requiring a high level of complexity in lab facilities and blood/plasma sampling, collection, and storage must occur under tightly controlled conditions. These demanding circumstances are especially difficult to attain in rural areas and in historically marginalized populations. The Telimmune Plasma Separation Card (TPS card or TPSC) has been developed to enable diagnostic plasma sampling, collection, and stabilization in locations that may be remote to laboratory or clinic. The TPSC requires a drop of blood applied to a top of a separation system consisting of a separation membrane and collection disk. In 3 min, the TPSC device separates plasma from erythrocytes and deposits a defined volume of plasma into a collection disc which is air-dried for 15 min to deliver a stabilized, volumetric plasma sample, which may be stored or shipped at ambient temperatures with minimal biological risk. Extraction of proteins and metabolites is then achieved in well-equipped laboratories using protocols discussed in this chapter., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryogenic electron microscopy (cryo-EM) is constantly developing and growing as a major technique for structure determination of protein complexes. Here, we detail the first steps of any cryo-EM project: specimen preparation and data collection. Step by step, a list of material needed is provided and the sequence of actions to carry out is given. We hope that these protocols will be useful to all people getting started with cryo-EM., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Molecular studies related to diagnosis and research rely on collection of blood samples and extraction of high-quality DNA. In Africa, where the populations carried 94% of the total burden of cases and deaths due to malaria in 2019, collection of samples is often challenged by remote study areas and lack of a cold chain to transport and store samples. Collection of blood on filter paper is a technique that is less invasive and has simpler requirements regarding training of staff, storage, and transport of samples than collection of venous blood samples. Dried blood spots (DBS) are therefore commonly used in many research projects. However, DNA quality can be affected by duration and conditions of storage. The quality of the DNA for molecular analyses also depends on a DNA extraction methodology that provides high-quality DNA with high purity and yield. Several protocols for DNA extraction have been described, and many comparative studies have analyzed and optimized the different methodologies to find an alternative to the more costly commercial extraction kits. This chapter describes recommendations for storage and preservation of DBS, and a Chelex-based protocol for extraction of DNA from DBS., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Arbuscular mycorrhizal fungi (AMF) are an important element of the plant microbiome as they establish an endosymbiotic relationship with the roots of most plant species. This association enhances access to nutrients and water for plants, and provides the fungus with plant-derived organic carbon. In this chapter, I describe a range of methods to work with AMF including: soil sampling; isolation of AMF propagules (spores, sporocarps, roots, and mycelium) by a wet sieving and centrifugation in a sucrose solution; trap (from field soil with AMF spores) and one-species pot cultures (from AMF spores divided into morphotypes); staining of mycorrhizae in plant roots; and production of diagnostic slides. These methods are widely used in taxonomic and ecological studies to characterize the morphology of AMF.

The study of fungal, bacterial, and other endophytic microorganisms using high throughput DNA sequencing requires sampling and processing of plant material that eliminates phylloplane microorganisms and retains those inside the plant compartment. Leaves, stems, roots, and other plant tissues are removed from the plant, washed, surface sterilized, and stored for downstream applications. Especially in ecological studies, field work for sample collection may take place in remote locations where laboratory equipment and resources are rudimentary, and accessing samples from target plants can be challenging. This chapter serves as a guide to basic protocols in the design and sample collection for studies focused on the endophytes of leaf, stem, and root tissues.

Quantitative PCR, if performed properly, is a highly sensitive and robust tool. Nevertheless, its application to the particular case of pathogen detection from foodstuffs necessitates special requirements for reliable results. Firstly, a robust analytical chain, involving sample preparation and DNA isolation with purification, is necessary to ensure optimal performance. Secondly, for reliable quantification of Listeria monocytogenes from food, reproducible controls for all steps of the analytical chain are needed, which can give quantitative information about the performance of each individual step of the detection chain. Ideally, each individual sample should include a so-called internal sample process control (ISPC) which passes through all steps of the analytical chain and is phenotypically similar to the target organism (in this case L. monocytogenes).This chapter describes the modular and rapid (3 h) sample preparation method "matrix lysis" for the quantification of L. monocytogenes from food and gives detailed information regarding the application of an ISPC based on the example of the L. monocytogenes Δ-prfA/+IAC strain.

Identification of molecular biomarkers for human diseases is one of the most important disciplines in translational science as it helps to elucidate their origin and early progression. Thus, it is a key factor in better diagnosis, prognosis, and treatment. Proteomics can help to solve the problem of sample complexity when the most common primary sample specimens were analyzed: organic fluids of easy access. The latest developments in high-throughput and label-free quantitative proteomics (SWATH-MS), together with more advanced liquid chromatography, have enabled the analysis of large sample sets with the sensitivity and depth needed to succeed in this task. In this chapter, we show different sample processing methods (major protein depletion, digestion, etc.) and a micro LC-SWATH-MS protocol to identify/quantify several proteins in different types of samples (serum/plasma, saliva, urine, tears).

In response to apparent damaging effects of plastics, especially microplastics, exposure to life, scientists have begun the arduous task of standardizing methods for the sample collection, separation, detection, and identification of microplastic particles. The ability to detect plastics depends upon the type of sample, procedure, instrument, expertise of the examiner, and the exact research question. The wide variability of sample processing and analyses does not lend itself well for cross-comparison of studies. However, with a multitude of procedures, techniques may be used in combination to successfully identify microplastic particles. Our goal in this chapter is not to provide a complete guide on plastic analyses, but to present an overview of the different sample collection, pretreatment, detection, and identification methodologies used for microplastic samples located in environmental and biological samples and to review advantages and limitations of each strategy.

In this chapter, we present an overview of a standard protocol to achieve structure determination at high resolution by Single Particle Analysis cryogenic Electron Microscopy using apoferritin as a standard sample. The purified apoferritin is applied to a glow-discharged support and then flash frozen in liquid ethane. The prepared grids are loaded into the electron microscope and checked for particle spreading and ice thickness. The microscope alignments are performed and the data collection session is setup for an overnight data collection. The collected movies containing two-dimensional images of the apoferritin sample are then processed to obtain a high-resolution three-dimensional reconstruction.

Outbreak analysis and transmission surveillance of viruses can be performed via whole-genome sequencing after viral isolation. Such techniques have recently been applied to characterize and monitor SARS-CoV-2 , the etiological agent of the COVID-19 pandemic. However, the isolation and culture of SARS-CoV-2 is time consuming and requires biosafety level 3 containment, which is not ideal for many resource-constrained settings. An alternate method, bait capture allows target enrichment and sequencing of the entire SARS-CoV-2 genome eliminating the need for viral culture. This method uses a set of hybridization probes known as "baits" that span the genome and provide sensitive, accurate, and minimal off-target hybridization. Baits can be designed to detect any known virus or bacteria in a wide variety of specimen types, including oral secretions. The bait capture method presented herein allows the whole genome of SARS-CoV-2 in saliva to be sequenced without the need to culture and provides an outline of bait design and bioinformatic analysis to guide a bioinformatician., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

Electron crystallography is a unique tool to study membrane protein structures and lipid-protein interactions in their native-like environments. Two-dimensional (2D) protein crystallization enables the lipids immobilized by the proteins, and the generated high-resolution density map allows us to model the atomic coordinates of the surrounding lipids to study lipid-protein interaction. This protocol describes the sample preparation for electron crystallographic studies, including back-injection method and carbon sandwich method. The protocols of data collection for electron crystallography, including electron imaging and diffraction, of the 2D membrane crystal will be followed.

Quantitative-PCR (qPCR) enables the quantification of specific DNA targets, such as functional or phylogenetic marker genes associated with environmental samples. During each qPCR cycle, the number of copies of a gene (or region) of interest in DNA samples is determined in real time using a fluorescence-based label and compared to a standard serial dilution. Here, we describe a qPCR method to quantify the ammonia oxidizing bacteria involved in the first step of nitrification, using the amoA gene as a proxy of their abundance. The preparation of the standards from environmental samples and qPCR is presented in detail for specifically quantifying microbial abundance in environmental samples such as soil.

Untargeted metabolomics has rapidly become a profiling method of choice in many areas of research, including mitochondrial biology. Most commonly, untargeted metabolomics is performed with liquid chromatography/mass spectrometry because it enables measurement of a relatively wide range of physiochemically diverse molecules. Specifically, to assess energy pathways that are associated with mitochondrial metabolism, hydrophilic interaction liquid chromatography (HILIC) is often applied before analysis with a high-resolution accurate mass instrument. The workflow produces large, complex data files that are impractical to analyze manually. Here, we present a protocol to perform untargeted metabolomics on biofluids such as plasma, urine, and cerebral spinal fluid with a HILIC separation and an Orbitrap mass spectrometer. Our protocol describes each step of the analysis in detail, from preparation of solvents for chromatography to selecting parameters during data processing.

Studying the plant phyllosphere to understand inhibition patterns to the growth of fungal foliar pathogens by using the Arabidopsis thaliana pathosystem offers unique opportunities for evaluating strategies for plant protection against foliar diseases. The wide array of bacteria inhabiting the phylloplane of plants has been researched to a much lesser extent compared to the bacteria in the rhizosphere. This difference is especially evident as bacteria derived from the aerial section of plants are rarely used in formulations of foliage sprays against pathogens and pests. In this chapter we outline easy and reliable methods for sample preparation to profile phyllosphere bacteria using high throughput amplicon sequencing and isolate/characterize potentially beneficial phyllosphere bacteria from Arabidopsis thaliana that inhibit in vitro the growth of foliar pathogens such as Alternaria brassicicola. The use of the described methods for profiling and screening phyllosphere bacteria may provide tangible progress on the discovery of new potential biological control agents against agriculturally important pathogens.

NMR spectroscopy has become one of the preferred analytical techniques for metabolomics studies due to its inherent nondestructive nature, ability to identify and quantify metabolites simultaneously in a complex mixture, minimal sample preparation requirement, and high degree of experimental reproducibility. NMR-based metabolomics studies involve the measurement and multivariate statistical analysis of metabolites present in biological samples such as biofluids, stool/feces, intestinal content, tissue, and cell extracts by high-resolution NMR spectroscopy-the goal then is to identify and quantify metabolites and evaluate changes of metabolite concentrations in response to some perturbation. Here we describe methodologies for NMR sample preparation of biofluids (serum, saliva, and urine) and stool/feces, intestinal content, and tissues for NMR experiments including extraction of polar metabolites and application of NMR in metabolomics studies. One dimensional (1D) 1 H NMR experiments with different variations such as pre-saturation, relaxation-edited, and diffusion-edited are routinely acquired for profiling and metabolite identification and quantification. 2D homonuclear 1 H- 1 H TOCSY and COSY, 2D J-resolved, and heteronuclear 1 H- 13 C HSQC and HMBC are also performed to assist with metabolite identification and quantification. The NMR data are then subjected to targeted and/or untargeted multivariate statistical analysis for biomarker discovery, clinical diagnosis, toxicological studies, molecular phenotyping, and functional genomics.

The vasculature of plants is typically colonized by a wide-range of bacteria with diverse functions. These bacteria can be sampled by pooling plant biopsies in water and then concentrating cells by centrifugation. When the extracted bacteria are added as a template for the polymerase chain reaction (PCR), sufficient DNA is generally liberated to facilitate the identification of specific taxa and characterization of bacterial community structure. The sampling technique facilitates surveys of multiple plants comprising a single crop, allowing for a more comprehensive understanding of the crop microbiome than what can be achieved when examining single plants. This technique is rapid and cost-effective, and will help researchers monitor microbes associated with vascular tissues at various stages of crop development.

Microcrystal Electron Diffraction (MicroED) enables structure determination of very small crystals that are much too small to be of use for other conventional diffraction techniques. MicroED has been used to determine the structures of many proteins and small organic molecules, and the technique can be performed on most standard cryo-TEM instruments equipped with high-speed detectors capable of collecting electron diffraction data. Here, we present protocols for MicroED sample preparation and data collection for protein microcrystals.

Detection of influenza A virus (IAV), viral antigen, nucleic acid, or antibodies in swine is dependent upon the collection of the appropriate specimen type, the quality of the specimen, and the proper storage and handling of the specimen. The diagnostic tests to be performed should be considered prior to specimen collection. Sera are acceptable specimens for ELISA or hemagglutination inhibition tests but not for real-time RT-PCR. Likewise, swabs, wipes, and/or tissues are acceptable for real-time RT-PCR and virus isolation. The specimen type will also depend on the age of the swine being tested; oral fluids can be successfully collected from weaned pigs usually greater than 3 weeks of age, whereas nasal or oral swabs should be collected from suckling pigs in the first weeks of life. The sensitivity of the RT-PCR test is such that IAV can be detected in not only the pig itself but also on surfaces that the pig contacts and in the air. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.

The 2016 Zika virus (ZIKV) outbreak in the Americas has been characterized by an increased association frequency of fetal neuropathological abnormalities. To have a comprehensive and accurate knowledge of key elements of the clinically observed neurologic dysfunctions in Zika-infected babies, ZIKV transmission from mother to fetus needs to be deeply studied. Thus, it is important to determine the role of both virus-targeted cells and tissues within the mother-fetus interface. Cellular tropism and mechanisms of ZIKV transmission from mother to the fetus during early pregnancy still remain unknown on many aspects. To improve the characterization of the maternal-fetal ZIKV transmission, we have set up an ex vivo model using an organ culture approach with a light-invasive sampling from the first trimester of pregnancy samples. Thus, here we provide evidence that circulating epidemic ZIKV strains from Latin America widely target and destroy reproductive tissues, including the decidua basalis, fetal placenta, and umbilical cord. In addition, we show that ZIKV is able to differentially replicate in a large range of both maternal and fetal cells, including decidual fibroblasts and macrophages, fetal trophoblast and Hofbauer cells, as well as umbilical cord mesenchymal stem cells. This primary and broad ZIKV cellular tropism and the resulting abundant cytopathic-induced tissue effects during the first trimester of pregnancy show the upstream path of clinically observed congenital damages.

Axon degeneration destructs functional connectivity of neural circuits and is one of the common, key pathological features of different neurodegenerative diseases. However, conventional histochemistry methods, which largely rely on tissue sections, have intrinsic limitations in examining the 3D distribution of axonal structures on the whole-tissue level. This technical shortcoming has continuously impeded our in-depth understanding of pathological axon degeneration in many scenarios. To overcome such drawback encountered in the research field, we describe here a general protocol of whole-tissue immunolabeling and 3D fluorescence imaging technique to visualize axon degeneration in the intact, unsectioned mouse tissues. In particular, experimental steps of tissue harvesting, whole-tissue immunolabeling, tissue optical clearing, and 3D fluorescence imaging have been systematically optimized, which makes the protocol effective for assessing integrity of the axonal structures in a variety of tissues. Notably, it has enabled the 3D fluorescence imaging of chemotherapy- or traumatic injury-induced axon degeneration within the bones (e.g., femurs) or bone-containing tissues (e.g., hindpaws), which had previously been inaccessible to conventional histochemistry methods. This protocol is therefore readily compatible with many areas of the research on axon degeneration and is poised to serve the field in future investigations.

Biobanking is important and fundamental for research and personalized medicine in patients with esophageal squamous cell carcinoma. The process often involves prospective collection of surgically obtained tissues (tissue banking) as well as serial blood samples (liquid biopsies) from the patients with esophageal squamous cell carcinoma. Apart from frozen tissues, formalin-fixed paraffin-embedded tissues are important sources of translational research. Careful planning and selection of the region of the paraffin-embedded tissues will maximize the use of tissue for molecular studies. Both cancer and non-cancer samples (controls) could be collected. The success and sustainability of the process needs proper infrastructure, advanced planning, funding, and multidisciplinary collaborations. The understanding of the principles and issues are detrimental for the success of biobanking. The technical procedures involved are standardized, complex, and time-consuming and needs coordinated taskforce.

In horses, presumptive diagnosis of equine influenza is commonly made on the basis of clinical signs. This alone is insufficient for confirmation of equine influenza, because other equine infectious respiratory diseases can in some degree have similar clinical presentations. Surveillance and control of equine influenza also necessitate detection of subclinical cases. Effective diagnosis of equine influenza virus infection is critically dependent on obtaining adequate specimens of virus-containing respiratory secretions for testing. These specimens are also valuable as sources for isolation of virus strains for antigenic characterization and potential inclusion in vaccines. Both nasal swabs and nasopharyngeal swabs are employed with horses. These differ little in their invasiveness, but nasopharyngeal swabs typically yield more virus than nasal swabs and are superior diagnostic specimens. Methods for obtaining nasopharyngeal swab specimens are described.

Histology, or the study of tissue microanatomy, is essential to understanding the in situ function of varying cell types within an organ. How cells are distributed throughout organs provides an indication of how they interact with other cells and structures within the organ microanatomy. The tortuous shape and large size of liver macrophages limits the value of standard tissue thickness of 5-10 μm. As a result, imaging of specimens ideally thicker than 100 μm is necessary to investigate the liver microanatomy and the how macrophages are distributed within this. Modern methods of microscopy, such as confocal and light sheet microscopy, allow for the analysis of tissue specimens of a thickness well beyond 100 μm in the z-dimension. Liver tissue is an opaque tissue, and as a result, different techniques are needed to ameliorate light diffraction within the tissue. These techniques, in conjunction with antibody staining and refractive index matching of the tissue, have allowed researchers to image liver tissue specimens of more than 100 μm thickness. Two of these techniques are modified versions of the clearing methods known as clearing-enhanced 3D (Ce3D) and fructose, urea, and glycerol for imaging (FUnGI). Here, we discuss the steps involved in preparing tissue specimens for optically clearing tissue using Ce3D and FUnGI for subsequent analysis of the distribution of macrophages in three dimensions using a confocal microscope.

Pathological assessment of frozen sections of tissues is important in the clinical management (intraoperative consultation) and research in patients with esophageal squamous cell carcinoma. Frozen sections may be used in the assessment of status of resection margins, extent of cancer metastasis (pathological staging), confirmation of the pathology, and increased volume of cancer cells for tissue banking. However, the applications of frozen sections have many technical limitations. Thus, interpretation of frozen sections needs expertise, collaborations, and attention to proper technical skills in the sectioning.

The ongoing development of single-particle cryo-electron microscopy (cryo-EM) is leading to fast data acquisition, data processing, and protein structure elucidation. Quick and reliable methods to go from protein purification and optimization to grid preparation will significantly improve the reach and power of cryo-EM. Such methods would particularly constitute a tremendous advantage in structural biology of membrane proteins, whose published structures stay still far behind the number of soluble protein structures. Here we describe a fast, low-cost, and user-friendly method for the purification and cryo-EM analysis of a recombinant membrane protein. This method minimizes the amount of starting material and manipulation steps needed to go from purification to grid preparation, and could potentially be expanded to other membrane protein purification systems for its direct application in structure determination by single-particle cryo-EM.

Cryo-electron microscopy (cryo-EM) is a powerful tool for investigating the structure of macromolecules under near-native conditions. Especially in the context of membrane proteins, this technique has allowed researchers to obtain structural information at a previously unattainable level of detail. Specimen preparation remains the bottleneck of most cryo-EM research projects, with membrane proteins representing particularly challenging targets of investigation due to their universal requirement for detergents or other solubilizing agents. Here we describe preparation of negative staining and cryo-EM grids and downstream data collection of membrane proteins in detergent, by far the most common solubilization agent. This protocol outlines a quick and straightforward procedure for screening and determining the structure of a membrane protein of interest under biologically relevant conditions.

Membrane interactions of proteins play a role in essential cellular processes in both physiological and disease states. The structural flexibility of intrinsically disordered proteins (IDPs) allows for interactions with multiple partners, including membranes. However, determining conformational states of IDPs when interacting with membranes can be challenging. Here we describe the use of nuclear magnetic resonance (NMR), including dark-state exchange saturation transfer (DEST), to probe IDP-membrane interactions in order to determine whether there is an interaction, which residues participate, and the extent/nature of the interaction between the protein and the membrane. Using α-synuclein and tau as typical examples, we provide protocols for how the membrane interactions of IDPs can be probed, including details of how the samples should be prepared and guidelines on how to interpret the results.

The efficient extraction and purification of viral RNA is critical for downstream molecular applications such as the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, gene sequencing, or quantification of avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types: enriched (e.g., virus stocks) and non-enriched (e.g., clinical). Clinical samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the extraction of AI virus RNA from common clinical specimen types and enriched virus stocks will be presented.

For tissues obtained from glioma samples with/without nonneoplastic brain there is no consensus for universal reference gene but there are some potential genes that might have good stability, under certain conditions. Considering all points described in this work, the care with tissue collection, until gene amplification, directly impacts on the reliable characterization of its mRNA levels. Moreover, it is clear the importance of selecting the most appropriate reference genes for each experimental situation, to allow the accurate normalization of target genes, especially for genes that are subtly regulated.

Wild birds are natural hosts of multiple microbial agents, including a wide diversity of coronaviruses. Here we describe a pan-Coronavirus detection RT-PCR method to identify those viruses regardless of the coronavirus genus or nature of the specimen. We also describe a protocol using high-throughput sequencing technologies to obtain their entire genome, which overcomes the inherent difficulties of wild bird coronavirus sequencing, that is, their genetic diversity and the lack of virus isolation methods.

Imaging mass spectrometry is a powerful technology that combines the molecular measurements of mass spectrometry with the spatial information inherent to microscopy. This unique combination of capabilities is ideally suited for the analysis of metabolites and lipids from single cells. This chapter describes a methodology for the sample preparation and analysis of single cells using high performance MALDI FTICR MS. Using this approach, we are able to generate profiles of lipid and metabolite expression from single cells that characterize cellular heterogeneity. This approach also enables the detection of variations in the expression profiles of lipids and metabolites induced by chemical stimulation of the cells. These results demonstrate that MALDI IMS provides an insightful view of lipid and metabolite expression useful in the characterization of a number of biological systems at the single cell level.

Individual developmental stages of blood fluke Schistosoma mansoni excrete or secrete a different set of molecules. Here we describe optimized protocols for collection of excretory/secretory products (E/S products) from cercariae, schistosomula, adult worms, and eggs. These E/S products are essential for successful parasitism functioning at the host-parasite interface, enabling invasion into the host and contributing to the survival of the parasite by modulation of host physiology and immune responses. Collection of sufficient amounts of E/S products is required for detailed research of these processes.

Tissue microarray (TMA) is widely used for identifying the expression of markers in many tissues from patients with esophageal squamous cell carcinoma. The technology is mostly used in immunohistochemical studies to test the expression of markers and oncoproteins in signalling pathway as well as targeting proteins involved in therapies for esophageal squamous cell carcinoma. Appropriate use of TMA sections needs consideration of labor, planning, and expertise involved. For the best performance, it is important to design the layout of the TMA as well as use whole-slide scanning for interpretation of the TMA sections.

Successful detection of avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel immunodiffusion tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs from gallinaceous poultry and cloacal swabs from waterfowl are the preferred specimens for most diagnostic tests, although it is optimal to collect swabs from both locations, if possible. In addition to collecting the appropriate sample for the tests to be performed, selecting the right materials for sample collection (i.e., type of swab) is very important. This chapter will outline the collection of different specimen types and procedures for proper specimen handling.

Basophils have been suggested to express low quantities of RNA, challenging the study of gene expression within these cells. However, the purification technique employed might have an impact on the quantity and quality of RNA purified from basophils. This chapter describes a method which gives an optimal RNA output using a TRIzol-based method in contrast to a commercial kit.

Whole-slide imaging (WSI) contributes to medical education, collaboration, quality assurance, examination, and consultation in pathology. The images obtained from WSI are of high quality and could be stored indefinitely. In research involving esophageal squamous cell carcinoma, the combination of WSI and image processing program allows effective interpretations of expressions of various immunomarkers related to pathogenesis, prognosis, and response to therapy in tissue microarray sections. The operation and basic principles of whole-slide imaging of esophageal squamous cell carcinoma are also presented. Common use of WSI will occur with modifications of the whole-slide imaging scanners to adapt to the workflows in diagnostic and research laboratories.

Chromosome analysis of chronic lymphocytic leukemia (CLL) is an important clinical tool for evaluating prognosis and disease progression. Visualizing chromosomes microscopically using traditional cytogenetic techniques requires dividing cells to be arrested during metaphase. The major challenge for performing this analysis on CLL samples is stimulating the cells to divide in culture. Stimulation of CLL cells with CpG oligodeoxynucleotides has improved our ability to perform chromosome analysis for this leukemia. This protocol should help the reader successfully culture CLL samples for clinical chromosome analysis.

Mass spectrometry (MS) proteomics allows systematic identification, characterization, and relative quantification of the full suite of proteins in a biological sample, and is a powerful analytical approach for investigation of many aspects of the biology of Neisseria meningitidis. Here, we describe methods for robust and efficient sample preparation of the proteome of N. meningitidis suitable for diverse MS proteomics workflows.

Transmission electron microscopy (TEM) is an ideal device to study the internal structure of cells and different types of biological materials, but adverse conditions inside electron microscopes such as damage induced by electron bombardment and vacuum evaporation of structural water necessitates complex preparation methods to survive this environment. In order to introduce the sample into the evacuated microscope column, it should be stabilized and altered to small enough (about 3 mm in diameter) and thin enough parts to permit the transmission of electrons. Depending on applications different thicknesses are required; for example, in biological research studies usually 300-500 nm thickness is indicated. To stabilize the specimen and preserve the sample structures, different preparation methods are used involving different steps based on the type of study and the specimen, although the ultimate goal of all these preparation technics is to maintain the native structure of the sample. In this chapter, we try to explain the series of steps that involve in preparation. Virtually every step can affect the quality of sample, and therefore it is important to execute each step in detail.

There is an important trend among museums and universities to digitize their collections both to help with archiving and allow remote access to their specimens (Olsen Museum specimens find new life online. The New York Times, 2015). While taxidermied animals, casts, and insect samples can be positioned and lit relatively easily, those that are preserved and stored in glass or Perspex jars need special lighting and a carefully thought out studio in order to get the best images. The photographs then need to be archived and stored to avoid loss. Many institutions are seeking to prepare 3D images, but this does not work for specimens contained with transparent vessels. In this chapter, we describe our approach to photography of fluid-preserved specimens.