Probes for INS

Spatial transcriptomics technologies are providing new insights to study gene expression, allowing researchers to investigate the spatial organization of transcriptomes in cells and tissues. This approach enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution, utilizing diverse technological approaches. In this perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques, as well as provide information on data analysis. In addition, this perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.

Visualizing and quantifying mRNA and its corresponding protein provides a unique perspective of gene expression at a single-molecule level. Here, we describe a method for differentiating primary cells for making airway epithelium and detecting SARS-CoV-2 Spike (S) mRNA and S protein in the paraformaldehyde-fixed paraffin-embedded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected airway epithelium. For simultaneous detection of mRNA and protein in the same cell, we combined two protocols: 1. RNA fluorescence-based in situ hybridization (RNA-FISH) based mRNA detection and 2. fluorescence-based immunohistochemistry (IHC) based protein detection. The detection of mRNA and proteins in the same cell also allows for quantifying them using the open-source software QuPath, which provides an accurate and more straightforward fluorescent-based quantification of mRNA and protein in the microscopic images of the infected cells. Additionally, we can achieve the subcellular distribution of both S mRNA and S protein. This method identifies SARS-CoV-2 S gene products' (mRNA and protein) degree of expression and their subcellular localization in the infected airway epithelium. Advantages of this method include: •Simultaneous detection and quantification of mRNA and protein in the same cell.•Universal use due to the ability to use mRNA-specific primer-probe and protein-specific antibodies.•An open-source software QuPath provides a straightforward fluorescent-based quantification.

Defining the cell-specific alternative splicing landscape in complex tissues is an important goal to gain functional insights. Deep-sequencing techniques coupled to genetic strategies for cell identification has provided important cues on cell-specific exon usage in complex tissues like the nervous system. BaseScope has emerged as a powerful and highly sensitive alternative to in situ hybridization to determine exon composition in tissue with spatial and morphological context. In this protocol, we will review how BaseScope was utilized to detect the e37a-Cacna1b splice variant of the presynaptic calcium channel CaV2.2 or N-type. This splice variant arises from a pair of mutually exclusive exons (e37a and e37b). E37a-Cacna1b is heavily underrepresented relative to e37b-Cacna1b and both exons share 60% of their sequence. By using BaseScope , we were able to discover that e37a-Cacna1b is expressed in excitatory pyramidal neurons of hippocampus and cortex, as well as motor neurons of the ventral horn of the spinal cord.

Since epigenetic modifications differ from cell to cell, detecting the DNA methylation status of individual cells is requisite. Therefore, it is important to conduct "morphology-based epigenetics research", in which the sequence-specific DNA methylation status is observed while maintaining tissue architecture. Here we demonstrate a novel histochemical technique that efficiently shows the presence of a single methylated cytosine in a sequence-dependent manner by applying ICON (interstrand complexation with osmium for nucleic acids) probes. By optimizing the concentration and duration of potassium osmate treatment, ICON probes selectively hybridize to methylated cytosine on tissue sections. Since the elongation process by rolling-circle amplification through the padlock probe and synchronous amplification by the hyperbranching reaction at a constant temperature efficiently amplifies the reaction, it is possible to specifically detect the presence of a single methylated cytosine. Since the ICON probe is cross-linked to the nuclear or mitochondrial DNA of the target cell, subsequent elongation and multiplication reactions proceed like a tree growing in soil with its roots firmly planted, thus facilitating the demonstration of methylated cytosine in situ. Using this novel ICON-mediated histochemical method, detection of the methylation of DNA in the regulatory region of the RANK gene in cultured cells and of mitochondrial DNA in paraffin sections of mouse cerebellar tissue was achievable. This combined ICON and rolling-circle amplification method is the first that shows evidence of the presence of a single methylated cytosine in a sequence-specific manner in paraffin sections, and is foreseen as applicable to a wide range of epigenetic studies.

The selection pressures that drove dramatic encephalisation processes through the mammal lineage remain elusive, as does knowledge of brain structure reorganisation through this process. In particular, considerable structural brain changes are present across the primate lineage, culminating in the complex human brain that allows for unique behaviours such as language and sophisticated tool use. To understand this evolution, a diverse sample set of humans' closest relatives with varying socio-ecologies is needed. However, current brain banks predominantly curate brains from primates that died in zoological gardens. We try to address this gap by establishing a field pipeline mitigating the challenges associated with brain extractions of wild primates in their natural habitat.The success of our approach is demonstrated by our ability to acquire a novel brain sample of deceased primates with highly variable socio-ecological exposure and a particular focus on wild chimpanzees. Methods in acquiring brain tissue from wild settings are comprehensively explained, highlighting the feasibility of conducting brain extraction procedures under strict biosafety measures by trained veterinarians in field sites.Brains are assessed at a fine-structural level via high-resolution MRI and state-of-the-art histology. Analyses confirm that excellent tissue quality of primate brains sourced in the field can be achieved with a comparable tissue quality of brains acquired from zoo-living primates.Our field methods are noninvasive, here defined as not harming living animals, and may be applied to other mammal systems than primates. In sum, the field protocol and methodological pipeline validated here pose a major advance for assessing the influence of socio-ecology on medium to large mammal brains, at both macro- and microstructural levels as well as aiding with the functional annotation of brain regions and neuronal pathways via specific behaviour assessments

Collection, preservation, and shipment of histological specimens in low-resource settings is challenging. We present a novel method that achieved excellent preservation of placental specimens from rural Mali by using formalin fixation, ethanol dehydration, and long-term storage in a solar-powered freezer. Sample preservation success was 92%, permitting evaluation of current and past malaria infection, anemia, placental maturity, and inflammation. Using RNAscope hybridization we were able to visualize cell-specific gene expression patterns in the formalin-fixed paraffin-embedded (FFPE) specimens. Additionally, our method entailed mirrored sampling from the two cut faces of a cotyledon, one for the FFPE workflows and the other for storage in RNAlater and RNA-seq.

The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.

In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.

This detailed volume collects commonly used and cutting-edge methods to analyze alternative splicing, a key step in gene regulation. After an introduction of the alternative splicing mechanism and its targeting for therapeutic strategies, the book continues with techniques for analyzing alternative splicing profiles in complex biological systems, visualizing and localizing alternative spliced transcripts with cellular and sub-cellular resolution, probing regulators of alternative splicing, as well as assessing the functional consequences of alternative splicing. Written for the highly successful _Methods in Molecular Biology_ series, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, reproducible protocols, and tips on troubleshooting and avoiding known pitfalls. 

In situ hybridization (ISH) is a powerful tool for investigating the spatial arrangement of nucleic acid targets in fixed samples. ISH is typically visualized using fluorophores to allow high sensitivity and multiplexing or with colorimetric labels to facilitate co-visualization with histopathological stains. Both approaches benefit from signal amplification, which makes target detection effective, rapid, and compatible with a broad range of optical systems. Here, we introduce a unified technical platform, termed 'pSABER', for the amplification of ISH signals in cell and tissue systems. pSABER decorates the in situ target with concatemeric binding sites for a horseradish peroxidase-conjugated oligonucleotide which can then catalyze the massive localized deposition of fluorescent or colorimetric substrates. We demonstrate that pSABER effectively labels DNA and RNA targets, works robustly in cultured cells and challenging formalin fixed paraffin embedded (FFPE) specimens. Furthermore, pSABER can achieve 25-fold signal amplification over conventional signal amplification by exchange reaction (SABER) and can be serially multiplexed using solution exchange. Therefore, by linking nucleic acid detection to robust signal amplification capable of diverse readouts, pSABER will have broad utility in research and clinical settings.

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAβ-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-βgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field.Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA.