A Novel Scoring System for MYB RNA In Situ Hybridization Displays High Sensitivity and Specificity for Adenoid Cystic Carcinoma in a Clinical Setting.

BACKGROUND: MYB RNA in situ hybridization (ISH) has emerged as a reliable and accessible marker to support adenoid cystic carcinoma (ACC) diagnosis, though still not well studied. Here, we report our results in a validation and prospective cohort to improve MYB RNA ISH diagnostic accuracy. METHODS: 79 cases (23 retrospective and 56 prospective) underwent MYB RNA ISH testing (44 ACC and 35 non-ACC). MYB RNA ISH results were initially interpreted based on previously established (original) scoring criteria. Weighted "i-scores", percent positive tumor cells, percent tumor cells with large signals (% LS), and staining pattern (abluminal, diffuse, focal non-patterned, or negative) were inputs for logistic regression models. Final model performance characteristics were compared with original scoring criteria and MYB::NFIB FISH results. RESULTS: An abluminal pattern was characteristic and exclusive to ACC. All i-scores, % LS, and percent positive were significantly higher in ACC. Original scoring criteria yielded a 95.5% sensitivity (Sn), 68.6% specificity (Sp), and 83.5% accuracy. MYB::NFIB FISH yielded a 42.9% sensitivity, 100% specificity, and 60% accuracy. Optimizing for performance, simplicity, and minimal collinearity, our final model was defined as: abluminal pattern and/or % LS > 16.5%, which resulted in a 93.2% Sn, 97.1% Sp, and 94.9% accuracy for ACC diagnosis. False negatives included an ACC with striking tubular eosinophilia and a MYBL1::NFIB translocated ACC. One false positive exclusive to the final model was a nasopharyngeal carcinoma with MYB amplification. CONCLUSIONS: MYB RNA ISH has a higher Sn than MYB::NFIB FISH while retaining high Sp. Our model provides improvements to specificity compared to original scoring criteria and highlight the importance of abluminal staining pattern and % LS. Nonetheless, alternate fusions remain key false negatives while rare non-ACC with other mechanisms of MYB activation may present as false positives.

Visualization of Individual RNA Molecules by Proximity Ligation-Based Chromogenic In Situ Hybridization Assay.

RNA in situ hybridization reveals the abundance and location of gene expression in cells or tissues, providing a technical basis for the clinical diagnosis of diseases. In this chapter, we show a "V" shape probe-mediated single-molecule chromogenic in situ hybridization (vsmCISH) technique for bright-field visualization of individual RNA molecules. In our method, several pairs of target hybridization probes are hybridized to RNA molecules and each probe pair forms a "V" shape overhang. The overhang oligonucleotides then mediated the proximity ligation to form DNA circles, followed by rolling circle amplification for signal enhancement and enzyme-catalyzed chromogenic reaction-based readout. The colorimetric assay avoids problems such as photobleaching and autofluorescence of current fluorescent in situ hybridization-based single-molecule RNA detection techniques. Furthermore, the relatively straightforward protocol makes the method useful for biological research and clinical diagnosis applications.

Improved methodology for fixation and preparation of Aedes aegypti embryos.

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100-150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies.

Effect of formalin-fixation and paraffin-embedded tissue storage times on RNAscope in situ hybridization signal amplification.

RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.

Gene Expression Patterns in the Ctenophore Pleurobrachia bachei: In Situ Hybridization.

In situ hybridization is a powerful and precise tool for revealing cell- and tissue-specific gene expression and a critical approach to validating single-cell RNA-seq (scRNA-seq). However, applying it to highly fragile animals such as ctenophores is challenging. Here, we present an in situ hybridization protocol for adult Pleurobrachia bachei (Cydippida)-a notable reference species representing the earliest-branching metazoan lineage, Ctenophora, sister to the rest of Metazoa. We provided expression patterns for several markers of cell phenotypes, as illustrated examples. The list includes predicted small secretory molecules/neuropeptides, WntX, genes encoding RNA-binding proteins (Musashi, Elav, Dicer, Argonaut), Neuroglobin, and selected transcription factors such as BarX. Both cell- and organ-specific expression of these genes further support the convergent evolution of many ctenophore innovations, which are remarkably distinct from tissue and organ specification in other basal metazoan lineages.

Coupling clearing and hybridization chain reaction approaches to investigate gene expression in organs inside intact insect heads.

Detecting RNA molecules within their natural environment inside intact arthropods has long been challenging, particularly in small organisms covered by a tanned and pigmented cuticle. Here, we have developed a methodology that enables high-resolution analysis of the spatial distribution of transcripts of interest without having to dissect tiny organs or tissues, thereby preserving their integrity. We have combined an in situ amplification approach based on hybridization chain reaction, which enhances the signal-to-noise ratio, and a clearing approach that allows the visualization of inner organs beneath the cuticle. We have implemented this methodology for the first time in Hemiptera, mapping two salivary aphid (Acyrthosiphon pisum) transcripts, the effector c002 and the salivary sheath protein SHP. With a multiplex approach, we could simultaneously detect different mRNAs in mounted pea aphid head-thorax samples and show that they were distributed in distinct secretory cells of salivary glands. RESEARCH HIGHLIGHTS: Combining hybridisation chain reaction and clearing allows the detection of transcripts in intact aphids heads. The transcripts of the two salivary proteins c002 and SHP are compartmentalized in distinct secretory cells of the principal glands.

Visualizing the translational activation of a particular mRNA in zebrafish embryos using in situ hybridization and proximity ligation assay.

Fertilized eggs initiate translation of stored mRNAs in spatially and temporally controlled manners. Here, we present a protocol for visualizing spatial and temporal translation in zebrafish embryos by fluorescence in situ hybridization and proximity ligation assay. We describe steps for labeling newly synthesized proteins and mRNA, visualizing mRNA translation and mRNA, sample mounting, and observation. Coupling detection of mRNA molecules with their translation sites is useful for understanding the molecular and cellular mechanisms that drive embryo development. For complete details on the use and execution of this protocol, please refer to Sato et al.1 and Takada et al.2.

CHRNA6 RNA In Situ Hybridization Is a Useful Tool for the Diagnosis of Extraskeletal Myxoid Chondrosarcoma.

Extraskeletal myxoid chondrosarcoma (EMC) is an uncommon mesenchymal neoplasm characteristically composed of uniform-appearing round to spindle-shaped cells with eosinophilic cytoplasm and abundant myxoid extracellular matrix. Although the majority of cases harbor a pathognomonic t(9;22) translocation that fuses EWSR1 with the orphan nuclear receptor NR4A3, there are less common variants that partner NR4A3 with TAF15, TCF12, or TFG. By immunohistochemistry, EMC has features of both cartilaginous and neuroendocrine differentiation, as evidenced by inconsistent expression of S100 protein and synaptophysin or INSM1, respectively, in a subset of cases. Given the limitations of available immunohistochemical stains for the diagnosis of EMC, we analyzed genome-wide gene expression microarray data to identify candidate biomarkers based on differential expression in EMC in comparison with other mesenchymal neoplasms. This analysis pointed to CHRNA6 as the gene with the highest relative expression in EMC (96-fold; P = 8.2 × 10-26) and the only gene with >50-fold increased expression in EMC compared with other tumors. Using RNA chromogenic in situ hybridization, we observed strong and diffuse expression of CHRNA6 in 25 cases of EMC, including both EWSR1-rearranged and TAF15-rearranged variants. All examined cases of histologic mimics were negative for CHRNA6 overexpression; however, limited CHRNA6 expression, not reaching a threshold of >5 puncta or 1 aggregate of chromogen in >25% of cells, was observed in 69 of 685 mimics (10.1%), spanning an array of mesenchymal tumors. Taken together, these findings suggest that, with careful interpretation and the use of appropriate thresholds, CHRNA6 RNA chromogenic in situ hybridization is a potentially useful ancillary histologic tool for the diagnosis of EMC.

Assessing the feasibility of a multimodal liquid biopsy for the diagnosis of HPV-associated oropharyngeal squamous cell carcinoma.

OBJECTIVES: In this feasibility study, we explored the combined use of circulating tumor human papillomavirus (HPV) DNA (ctHPVDNA) and HPV serology as diagnostic tests for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). METHODS: Among patients with research-banked serum or plasma at diagnosis, IgG antibodies to oncoproteins from HPV types 16, 18, 31, 33, 35, 45, 52, and 58 were detected with multiplex serology. Positivity for HPV 16 was defined based on detection of combinations of anti-E6, E1, E2, and E7 and for other high-risk types on detection of anti-E6 and anti-E7. Circulating tumor HPV DNA was detected by custom digital droplet polymerase chain reaction (ddPCR) assays for HPV types 16, 18, 33, 35, and 45. p16 immunohistochemistry and high-risk HPV RNA in situ hybridization (ISH) using a cocktail of 18 high-risk HPV types were performed on tissue. RESULTS: Of 75 patients, 67 (89.3%) were HPV-associated (p16 and HPV RNA ISH positive) and 8 (10.7%) were HPV-independent. All 8 HPV-independent patients were seronegative and negative for ctHPVDNA (100% specificity). Serology was positive in 53 (79.1%) of 67 HPV-associated patients, while ddPCR was positive for ctHPVDNA in 59 (88.6%) of 67 HPV-associated patients. Requiring both tests to be positive resulted in a sensitivity of 50 (74.6%) of 67 while combining assays (either positive) improved sensitivity to 62 (92.6%) of 67. CONCLUSIONS: Compared to HPV RNA ISH, HPV serology and ctHPVDNA are sensitive and highly specific biomarkers for HPV-associated OPSCC at the time of presentation.

Molecular Characterization, Expression and In Situ Hybridization Analysis of a Pedal Peptide/Orcokinin-type Neuropeptide in Cuttlefish Sepiella japonica.

BACKGROUND: Neuropeptide pedal peptide (PP) and orcokinin (OK), which are structurally related active peptides, have been widely discovered in invertebrates and constitute the PP/OK neuropeptide family. They have complex structures and play myriad roles in physiological processes. To date, there have been no related reports of PP/OK-type neuropeptide in cephalopods, which possess a highly differentiated multi-lobular brain. METHODS: Rapid Amplification of cDNA Ends (RACE) was employed to obtain the open reading frame (ORF) of PP/OK-type neuropeptide in Sepiella japonica (termed as Sj-PP/OK). Various software were used for sequence analysis. Semi-quantitative PCR was applied to analyze the tissue distribution profile, quantitative real-time PCR (qRT-PCR) was used to study spatio-temporal expression throughout the entire growth and development period, and in situ hybridization (ISH) was employed to observe the tissue location of Sj-PP/OK. RESULTS: in the present study, we identified the ORF of Sj-PP/OK. The putative precursor of Sj-PP/ OK encodes 22 mature peptides, of which only tridecapeptides could undergo post-translationally amidated at C-terminus. Each of these tridecapeptides possesses the most conserved and frequent N-terminus Asp-Ser-Ile (DSI). Sequence analysis revealed that Sj-PP/OK shared comparatively low identity with other invertebrates PP or OK. The tissue distribution profile showed differences in the expression level of Sj-PP/OK between male and female. qRT-PCR data demonstrated that Sj-PP/OK was widely distributed in various tissues, with its expression level increasing continuously in the brain, optic lobe, liver, and nidamental gland throughout the entire growth and development stages until gonad maturation. ISH detected that Sj-PP/OK positive signals existed in almost all regions of the optic lobe except the plexiform zone, the outer edge of all functional lobes in the brain, epithelial cells and the outer membrane layer of the accessory nidamental gland. These findings suggest that Sj-PP/OK might play a role in the regulation of reproduction, such as vitellogenin synthesis, restoration, and ova encapsulation. CONCLUSION: The study indicated that Sj-PP/OK may be involved in the neuroendocrine regulation in cephalopods, providing primary theoretical basis for further studies of its regulation role in reproduction.

Variable levels of spike and ORF1ab RNA in post-mortem lung samples of SARS-CoV-2-positive subjects: comparison between ISH and RT-PCR.

Post-mortem examination plays a pivotal role in understanding the pathobiology of the SARS-CoV-2; thus, the optimization of virus detection on the post-mortem formalin-fixed paraffin-embedded (FFPE) tissue is needed. Different techniques are available for the identification of the SARS-CoV-2, including reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy. The main goal of this study is to compare ISH versus RT-PCR to detect SARS-CoV-2 on post-mortem lung samples of positive deceased subjects. A total of 27 samples were analyzed by RT-PCR targeting different viral RNA sequences of SARS-CoV-2, including envelope (E), nucleocapsid (N), spike (S), and open reading frame (ORF1ab) genes and ISH targeting S and Orf1ab. All 27 cases showed the N gene amplification, 22 out of 27 the E gene amplification, 26 out of 27 the S gene amplification, and only 6 the ORF1ab gene amplification. The S ISH was positive only in 12 out of 26 cases positive by RT-PCR. The S ISH positive cases with strong and diffuse staining showed a correlation with low values of the number of the amplification cycles by S RT-PCR suggesting that ISH is a sensitive assay mainly in cases carrying high levels of S RNA. In conclusion, our findings demonstrated that ISH assay has lower sensitivity to detect SARS-CoV-2 in FFPE compared to RT-PCR; however, it is able to localize the virus in the cellular context since it preserves the morphology.

Djhsp70s, especially Djhsp70c, play a key role in planarian regeneration and tissue homeostasis by regulating cell proliferation and apoptosis.

Heat shock protein 70 family (HSP70s) is one of the most conserved and important group of HSPs as molecular chaperones, which plays an important role in cytoprotection, anti-apoptosis and so on. However, the molecular mechanism of HSP70s in animal regeneration remains to be delineated. In this study, we investigate the roles of HSP70s in regeneration of planarian. The four genes, Djhsp70a, Djhsp70b, Djhsp70c, and Djhsp70d of the HSP70s, are selected from the transcriptome database, because of their high expression levels in planarians. We then study the biological roles of each gene by conducting various experimental techniques, including RNAi, RT-PCR, WISH, Whole-mount immunostaining and TUNEL. The results show: (1) External stressors, such as temperature, tissue damage and ionic liquid upregulate the expression of Djhsp70s significantly. (2) The gene expression of Djhsp70s in planarians exhibits dynamic patterns. According to the result of WISH, the Djhsp70s are mainly expressed in parenchymal tissues on both sides of the body as well as blastema. It is consistent with the data of qRT-PCR. (3) After RNA interference of Djhsp70s, the worms experience cephalic regression and lysis, body curling, stagnant regeneration and death. (4) Knockdown of Djhsp70s affect the cell proliferation and apoptosis. These results suggest that Djhsp70s are not only conserved in cytoprotection, but involved in homeostasis maintenance and regeneration process by regulating coordination of cell proliferation and apoptosis in planarians.

Hemoglobin in the blood acts as a chemosensory signal via the mouse vomeronasal system.

The vomeronasal system plays an essential role in sensing various environmental chemical cues. Here we show that mice exposed to blood and, consequently, hemoglobin results in the activation of vomeronasal sensory neurons expressing a specific vomeronasal G protein-coupled receptor, Vmn2r88, which is mediated by the interaction site, Gly17, on hemoglobin. The hemoglobin signal reaches the medial amygdala (MeA) in both male and female mice. However, it activates the dorsal part of ventromedial hypothalamus (VMHd) only in lactating female mice. As a result, in lactating mothers, hemoglobin enhances digging and rearing behavior. Manipulation of steroidogenic factor 1 (SF1)-expressing neurons in the VMHd is sufficient to induce the hemoglobin-mediated behaviors. Our results suggest that the oxygen-carrier hemoglobin plays a role as a chemosensory signal, eliciting behavioral responses in mice in a state-dependent fashion.

Sensitive visualization of SARS-CoV-2 RNA with CoronaFISH.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The positive-sense single-stranded RNA virus contains a single linear RNA segment that serves as a template for transcription and replication, leading to the synthesis of positive and negative-stranded viral RNA (vRNA) in infected cells. Tools to visualize vRNA directly in infected cells are critical to analyze the viral replication cycle, screen for therapeutic molecules, or study infections in human tissue. Here, we report the design, validation, and initial application of FISH probes to visualize positive or negative RNA of SARS-CoV-2 (CoronaFISH). We demonstrate sensitive visualization of vRNA in African green monkey and several human cell lines, in patient samples and human tissue. We further demonstrate the adaptation of CoronaFISH probes to electron microscopy. We provide all required oligonucleotide sequences, source code to design the probes, and a detailed protocol. We hope that CoronaFISH will complement existing techniques for research on SARS-CoV-2 biology and COVID-19 pathophysiology, drug screening, and diagnostics.

Evaluation of deep convolutional neural networks for in situ hybridization gene expression image representation.

High resolution in situ hybridization (ISH) images of the brain capture spatial gene expression at cellular resolution. These spatial profiles are key to understanding brain organization at the molecular level. Previously, manual qualitative scoring and informatics pipelines have been applied to ISH images to determine expression intensity and pattern. To better capture the complex patterns of gene expression in the human cerebral cortex, we applied a machine learning approach. We propose gene re-identification as a contrastive learning task to compute representations of ISH images. We train our model on an ISH dataset of ~1,000 genes obtained from postmortem samples from 42 individuals. This model reaches a gene re-identification rate of 38.3%, a 13x improvement over random chance. We find that the learned embeddings predict expression intensity and pattern. To test generalization, we generated embeddings in a second dataset that assayed the expression of 78 genes in 53 individuals. In this set of images, 60.2% of genes are re-identified, suggesting the model is robust. Importantly, this dataset assayed expression in individuals diagnosed with schizophrenia. Gene and donor-specific embeddings from the model predict schizophrenia diagnosis at levels similar to that reached with demographic information. Mutations in the most discriminative gene, Sodium Voltage-Gated Channel Beta Subunit 4 (SCN4B), may help understand cardiovascular associations with schizophrenia and its treatment. We have publicly released our source code, embeddings, and models to spur further application to spatial transcriptomics. In summary, we propose and evaluate gene re-identification as a machine learning task to represent ISH gene expression images.

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Advanced detection strategies for cardiotropic virus infection in a cohort study of heart failure patients.

The prevalence and contribution of cardiotropic viruses to various expressions of heart failure are increasing, yet primarily underappreciated and underreported due to variable clinical syndromes, a lack of consensus diagnostic standards and insufficient clinical laboratory tools. In this study, we developed an advanced methodology for identifying viruses across a spectrum of heart failure patients. We designed a custom tissue microarray from 78 patients with conditions commonly associated with virus-related heart failure, conditions where viral contribution is typically uncertain, or conditions for which the etiological agent remains suspect but elusive. Subsequently, we employed advanced, highly sensitive in situ hybridization to probe for common cardiotropic viruses: adenovirus 2, coxsackievirus B3, cytomegalovirus, Epstein-Barr virus, hepatitis C and E, influenza B and parvovirus B19. Viral RNA was detected in 46.4% (32/69) of heart failure patients, with 50% of virus-positive samples containing more than one virus. Adenovirus 2 was the most prevalent, detected in 27.5% (19/69) of heart failure patients, while in contrast to previous reports, parvovirus B19 was detected in only 4.3% (3/69). As anticipated, viruses were detected in 77.8% (7/9) of patients with viral myocarditis and 37.5% (6/16) with dilated cardiomyopathy. Additionally, viruses were detected in 50% of patients with coronary artery disease (3/6) and hypertrophic cardiomyopathy (2/4) and in 28.6% (2/7) of transplant rejection cases. We also report for the first time viral detection within a granulomatous lesion of cardiac sarcoidosis and in giant cell myocarditis, conditions for which etiological agents remain unknown. Our study has revealed a higher than anticipated prevalence of cardiotropic viruses within cardiac muscle tissue in a spectrum of heart failure conditions, including those not previously associated with a viral trigger or exacerbating role. Our work forges a path towards a deeper understanding of viruses in heart failure pathogenesis and opens possibilities for personalized patient therapeutic approaches.

NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1. In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH.

Step-by-step preparation of mouse eye sections for routine histology, immunofluorescence, and RNA in situ hybridization multiplexing.

It can be challenging to maintain tissue integrity using established histology protocols. Here, we describe a protocol composed of Hartman's fixation, window technique, microwave-based tissue processing, optimized depigmentation, and antigen retrieval pretreatment. This is followed by the ViewRNA single-molecule fluorescence in situ hybridization and immunofluorescence techniques to optimize routine histological staining and molecular histology multiplexing assays. Our protocol is highly reproducible in any laboratory and may decrease animal usage and lab resource expenditure. For complete details on the use and execution of this protocol, please refer to Pang et al. (2021).