Probes for INS

Gene expression analyses by molecular histology are a crucial step in understanding gene function in any model organism. In the teleost _Astyanax mexicanus_, here we describe in detail the method we have developed to perform double fluorescent in situ hybridization on whole-mount samples. As an illustration, in the result section, we present an analysis of the expression patterns of four mRNAs expressed in the hypothalamus of the surface and cave morphs of _A. mexicanus_ at 3.5 days postfertilization, three neuropeptides (_npy_, _pomca_, _agrp_) and one transcription factor (_isl1_). Confocal imaging after fluorescent in situ hybridization allows counting cells in distinct but closely related hypothalamic areas. The step-by-step protocol and the comprehensive table of reagents presented here will allow researchers to analyze gene expression in different structures and at various stages, from embryos to older larvae.

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Radiochemical in situ hybridization enables detection of gene expression in small areas of the brain, such as the developing pineal gland in rodents. The method combines determination of spatial and temporal gene expression profiles with semiquantitative analyses. We here describe the procedure of radiochemical in situ hybridization on the developing rat pineal gland ranging from preparation of fetal tissue for in situ hybridization to principles of quantification.

Single-cell technology has become an indispensable tool in cardiovascular research since its first introduction in 2009. Here, we highlight the recent remarkable progress in using single-cell technology to study transcriptomic and epigenetic heterogeneity in cardiac disease and development. We then introduce the key concepts in single-cell multi-omics modalities that apply to cardiovascular research. Lastly, we discuss some of the trending concepts in single-cell technology that are expected to propel cardiovascular research to the next phase of single-cell research.

Epidermal keratinocytes express various purinergic 2 receptors that play an essential role in cell growth, differentiation, and proliferation. In the conditions of injury, concentrations of extracellular adenosine triphosphate (ATP) may dramatically increase due to cell damage and inflammatory processes. In this situation activation of purinergic signaling in keratinocytes could act as a double-edged sword contributing to skin regeneration or cell apoptosis. As the role of keratinocytes in transducing and modulating nociceptive stimuli has been increasingly appreciated in recent years, the aim of the present study was to evaluate whether peripheral nerve injury affects purinergic signaling in keratinocytes. Spared nerve injury (SNI), a classical model of peripheral neuropathic pain, was induced in mice. The injury was induced by sparing of the tibial nerve, and ligation and cut of the sural and common peroneal nerves. Keratinocytes were isolated and cultured on Days 2-4 post-injury and ATP-mediated calcium responses in keratinocytes were examined by confocal imaging. On average, the number of keratinocytes that responded to ATP with an increase in intracellular calcium gradient as well as the magnitude of the peak response was not significantly different between sham and SNI groups. However, significantly less delay in ATP-induced increase in intracellular calcium concentration was observed in keratinocytes in SNI group compared to sham. Selective pharmacological inhibition of keratinocyte response to ATP indicated a major role of P2 × 4 receptors in the modulation of calcium homeostasis in SNI. Our results indicate that epidermal purinergic signaling undergoes dramatic changes following peripheral nerve injury that may contribute to injury-induced mechanical hypersensitivity.

Localization of mRNAs at the front of migrating cells is a widely used mechanism that functionally supports efficient cell movement. It is observed in single cells on two-dimensional surfaces, as well as in multicellular three-dimensional (3D) structures and in tissue in vivo. 3D multicellular cultures can reveal how the topology of the extracellular matrix and cell-cell contacts influence subcellular mRNA distributions. Here we describe a method for mRNA imaging in an inducible system of collective cancer cell invasion. MDA-MB-231 cancer cell spheroids are embedded in Matrigel, induced to invade, and processed to image mRNAs with single-molecule sensitivity. An analysis algorithm is used to quantify and compare mRNA distributions at the front of invasive leader cells. The approach can be easily adapted and applied to analyze RNA distributions in additional settings where cells polarize along a linear axis.

Chronic stress is a well-known risk factor for the development of hypertension. However, the underlying mechanisms remain unclear. Corticotropin-releasing hormone (CRH) neurons in the central nucleus of the amygdala (CeA) are involved in the autonomic responses to chronic stress. Here, we determined the role of CeA-CRH neurons in chronic stress-induced hypertension.Borderline hypertensive rats (BHRs) and Wistar-Kyoto (WKY) rats were subjected to chronic unpredictable stress (CUS). Firing activity and M-currents of CeA-CRH neurons were assessed, and a CRH-Cre-directed chemogenetic approach was used to suppress CeA-CRH neurons. CUS induced a sustained elevation of arterial blood pressure (ABP) and heart rate (HR) in BHRs, while in WKY rats, CUS-induced increases in ABP and HR quickly returned to baseline levels after CUS ended. CeA-CRH neurons displayed significantly higher firing activities in CUS-treated BHRs than unstressed BHRs. Selectively suppressing CeA-CRH neurons by chemogenetic approach attenuated CUS-induced hypertension and decreased elevated sympathetic outflow in CUS-treated BHRs. Also, CUS significantly decreased protein and mRNA levels of Kv7.2 and Kv7.3 channels in the CeA of BHRs. M-currents in CeA-CRH neurons were significantly decreased in CUS-treated BHRs compared with unstressed BHRs. Blocking Kv7 channel with its blocker XE-991 increased the excitability of CeA-CRH neurons in unstressed BHRs but not in CUS-treated BHRs. Microinjection of XE-991 into the CeA increased sympathetic outflow and ABP in unstressed BHRs but not in CUS-treated BHRs.CeA-CRH neurons are required for chronic stress-induced sustained hypertension. The hyperactivity of CeA-CRH neurons may be due to impaired Kv7 channel activity, which represents a new mechanism involved in chronic stress-induced hypertension.We found that hyperactivity of CRH neurons in the CeA, likely due to diminished Kv7 channel activity, play a major role in the development of chronic stress-induced hypertension. Our study suggests that CRH neurons in the brain may be targeted for treating chronic stress-induced hypertension. Thus, increasing Kv7 channel activity or overexpressing Kv7 channels in the CeA may reduce stress-induced hypertension. Further studies are needed to delineate how chronic stress diminishes Kv7 channel activity in the brain.

Somatosensory neurons are highly heterogeneous with distinct types of neural cells responding to specific stimuli. However, the distribution and roles of cell-type-specific long intergenic noncoding RNAs (lincRNAs) in somatosensory neurons remain largely unexplored. Here, by utilizing droplet-based single-cell RNA-seq (scRNA-seq) and full-length Smart-seq2, we show that lincRNAs, but not coding mRNAs, are enriched in specific types of mouse somatosensory neurons. Profiling of lincRNAs from single neurons located in dorsal root ganglia (DRG) identifies 200 lincRNAs localized in specific types or subtypes of somatosensory neurons. Among them, the conserved cell-type-specific lincRNA CLAP associates with pruritus and is abundantly expressed in somatostatin (SST)-positive neurons. CLAP knockdown reduces histamine-induced Ca2+ influx in cultured SST-positive neurons and in vivo reduces histamine-induced scratching in mice. In vivo knockdown of CLAP also decreases the expression of neuron-type-specific and itch-related genes in somatosensory neurons, and this partially depends on the RNA binding protein MSI2. Our data reveal a cell-type-specific landscape of lincRNAs and a function for CLAP in somatosensory neurons in sensory transmission.

Microglia are widely distributed in the central nervous system (CNS), where they aid in the maintenance of neuronal function and perform key auxiliary roles in phagocytosis, neural repair, immunological control, and nutrition delivery. Microglia in the undamaged spinal cord is in a stable state and serve as immune monitors. In the event of spinal cord injury (SCI), severe changes in the microenvironment and glial scar formation lead to axonal regeneration failure. Microglia participates in a series of pathophysiological processes and behave both positive and negative consequences during this period. A deep understanding of the characteristics and functions of microglia can better identify therapeutic targets for SCI. Technological innovations such as single-cell RNA sequencing (Sc-RNAseq) have led to new advances in the study of microglia heterogeneity throughout the lifespan. Here,We review the updated studies searching for heterogeneity of microglia from the developmental and pathological state, survey the activity and function of microglia in SCI and explore the recent therapeutic strategies targeting microglia in the CNS injury.

Introduction: Pulmonary arterial hypertension (PAH) is a lethal condition lacking curative pharmacotherapy. Expansion of the extracellular matrix occurs in early stages of pulmonary angiopathy, but the presence of individual matrix components warrants further investigation. Accumulation of the osmotically active matrix proteoglycan aggrecan has been associated with swelling and disruption of vessel wall integrity in systemic arteries. Whether aggrecan is present to any significant extent in PAH tissue, and what potential role it may have, is not known. Methods: Paraffin-embedded lung tissue from 11 patients with idiopathic PAH was imaged using synchrotron-based phase contrast micro-CT at the TOMCAT beamline, Swiss Light Source. Image analysis was performed in Fiji and Amira. Imaged blocks were subsequently sectioned for histology, immunohistochemistry with an aggrecan core protein antibody and RNAscope in situ hybridization. qPCR was performed to investigate gene expression. Failed donor lungs were used as controls. Results: Aggrecan core protein was identified in vascular lesions of all 11 patients with idiopathic PAH, localized to cellular rather than fibrotic or collagenous lesions. RNAscope in situ hybridization confirmed local production of ACAN mRNA in diseased vessels. Quantification of repeated immunohistochemistry demonstrated significantly increased accumulation of aggrecan in patients with idiopathic PAH compared to failed donor lung controls. ACAN and ADAMTS15 mRNA were also found to be up-regulated in pulmonary arteries from patients with IPAH, indicating ongoing proteolytic turnover. Image analysis and three-dimensional renderings of pulmonary arteries identified aggrecan in lumen-reducing lesions containing cellular connective tissue, at sites of intrapulmonary bronchopulmonary shunting and at sites of elevated pulmonary blood pressure. Conclusions: Our findings indicate local production and accumulation of aggrecan in pressure-related lesions of idiopathic PAH. This work strengthens the hypothesis that aggrecan plays a role in arterial adaptations to altered hemodynamics and is the first to suggest a role for aggrecan in pulmonary arterial homeostasis and idiopathic PAH.

Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies. Using ACD's integrated Co-Detection Workflow (ICW), we have developed a protocol to use phospho-specific antibodies in combination with ISH to show co-localization of EGFR mRNA and EGFR proteins phosphorylated at different sites in tumor cells. Our protocol has been used for multiplexing Y1086 phosphorylated EGFR, Y1068 phosphorylated EGFR, and EGFR RNA in A431 human epidermoid carcinoma cells.

The etiology of bladder cancer is known to be associated with behavioral and environmental factors. Moreover, several studies suggested a potential role of HPV infection in the pathogenesis with controversial results. A systematic review was conducted to assess the role of HPV. A total of 46 articles that reported the prevalence of HPV infection in squamous (SCC), urothelial (UC), and transitional cell carcinomas (TCC) were selected. A pooled prevalence of 19% was found, with a significant difference in SCC that was mainly driven by HPV-16. Moreover, infection prevalence in case-control studies showed a higher risk of bladder cancer in HPV-positive cases (OR: 7.84; p-value < 0.00001). The results may suggest an etiologic role of HPV in bladder cancer. HPV vaccine administration in both sexes could be key to prevent the infection caused by high-risk genotypes.