Comprehensive mapping of histamine H1 receptor mRNA in the mouse brain.

Histamine H1 receptor (H1R) in the central nervous system plays an important role in various functions, including learning and memory, aggression, feeding behaviors, and wakefulness, as evidenced by studies utilizing H1R knockout mice and pharmacological interventions. Although previous studies have reported the widespread distribution of H1R in the brains of rats, guinea pigs, monkeys, and humans, the detailed distribution in the mouse brain remains unclear. This study provides a comprehensive description of the distribution of H1R mRNA in the mouse brain using two recently developed techniques: RNAscope and in situ hybridization chain reaction, both of which offer enhanced sensitivity and resolution compared to traditional methodologies such as radioisotope labeling, which were used in previous studies. The H1R mRNA expression was observed throughout the entire brain, including key regions implicated in sleep-wake regulatory functions, such as the pedunculopontine tegmental nucleus and dorsal raphe. Additionally, strong H1R mRNA signals were identified in the paraventricular hypothalamus and ventromedial hypothalamus, which may explain the potential mechanisms underlying histamine-mediated feeding regulation. Notably, we identified strong H1R mRNA expression in previously unreported cerebral regions, such as the dorsal endopiriform nucleus, bed nucleus of the accessory olfactory tract, and postsubiculum. These findings significantly contribute to our understanding of the multifaceted roles of H1R in diverse brain functions.

Correlation of TRPA1 RNAscope and Agonist Responses.

The TRPA1 ion channel is a sensitive detector of reactive chemicals, found primarily on sensory neurons. The phenotype exhibited by mice lacking TRPA1 suggests its potential as a target for pharmacological intervention. Antibody-based detection for distribution analysis is a standard technique. In the case of TRPA1, however, there is no antibody with a plausible validation in knockout animals or functional studies, but many that have failed in this regard. To this end we employed the single molecule in situ hybridization technique RNAscope on sensory neurons immediately after detection of calcium responses to the TRPA1 agonist allyl isothiocyanate. There is a clearly positive correlation between TRPA1 calcium imaging and RNAscope detection (R = 0.43), although less than what might have been expected. Thus, the technique of choice should be carefully considered to suit the research question. The marginal correlation between TRPV1 RNAscope and the specific agonist capsaicin indicates that such validation is advisable for every RNAscope target. Given the recent description of a long-awaited TRPA1 reporter mouse, TRPA1 RNAscope detection might still have its use cases, for detection of RNA at particular sites, for example, defined structurally or by other molecular markers.

ENAM Mutations Can Cause Hypomaturation Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a diverse group of inherited diseases featured by various presentations of enamel malformations that are caused by disturbances at different stages of enamel formation. While hypoplastic AI suggests a thickness defect of enamel resulting from aberrations during the secretory stage of amelogenesis, hypomaturation AI indicates a deficiency of enamel mineralization and hardness established at the maturation stage. Mutations in ENAM, which encodes the largest enamel matrix protein, enamelin, have been demonstrated to cause generalized or local hypoplastic AI. Here, we characterized 2 AI families with disparate hypoplastic and hypomaturation enamel defects and identified 2 distinct indel mutations at the same location of ENAM, c588+1del and c.588+1dup. Minigene splicing assays demonstrated that they caused frameshifts and truncation of ENAM proteins, p.Asn197Ilefs*81 and p.Asn197Glufs*25, respectively. In situ hybridization of Enam on mouse mandibular incisors confirmed its restricted expression in secretory stage ameloblasts and suggested an indirect pathogenic mechanism underlying hypomaturation AI. In silico analyses indicated that these 2 truncated ENAMs might form amyloid structures and cause protein aggregation with themselves and with wild-type protein through the added aberrant region at their C-termini. Consistently, protein secretion assays demonstrated that the truncated proteins cannot be properly secreted and impede secretion of wild-type ENAM. Moreover, compared to the wild-type, overexpression of the mutant proteins significantly increased endoplasmic reticulum stress and upregulated the expression of unfolded protein response (UPR)-related genes and TNFRSF10B, a UPR-controlled proapoptotic gene. Caspase, terminal deoxynucleotidyl transferase UTP nick-end labeling (TUNEL), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays further revealed that both truncated proteins, especially p.Asn197Ilefs*81, induced cell apoptosis and decreased cell survival, suggesting that the 2 ENAM mutations cause AI through ameloblast cell pathology and death rather than through a simple loss of function. This study demonstrates that an ENAM mutation can lead to generalized hypomaturation enamel defects and suggests proteinopathy as a potential pathogenesis for ENAM-associated AI.

Knockdown of vitamin D receptor affects early stages of pectoral fin development in zebrafish.

The vitamin D receptor (VDR) signalling has been implicated in vertebrate limb or fin formation. However, the involvement of VDR signalling in the early stages of limb/fin development remains to be elucidated. In this study, the role of VDR signalling in pectoral fin development was investigated in zebrafish embryos. Knockdown of vdr induced the severe impairment of pectoral fin development. The zebrafish larvae lacking vdr exhibited reduced pectoral fins with no skeletal elements. In situ hybridization revealed depletion of vdr downregulated fibroblast growth factor 24 (fgf24), a marker of early pectoral fin bud mesenchyme, in the presumptive fin field even before fin buds were visible. Moreover, a perturbed expression pattern of bone morphogenetic protein 4 (bmp4), a marker of the pectoral fin fold, was observed in the developing fin buds of zebrafish embryos that lost the vdr function. These findings suggest that VDR signalling is crucial in the early stages of fin development, potentially influencing the process by regulating other signalling molecules such as Fgf24 and Bmp4.

EBV-Associated Gastric Cancer; An In Situ Hybridization Assay on Tissue Microarray: A Multi-Region Study from Four Major Provinces of Iran.

BACKGROUND: Gastric cancer is the fourth leading cause of cancer-related deaths in the world. The identification of gastric cancer subtypes related to recognizable microbial agents may play a pivotal role in the targeted prevention and treatment of this cancer. The current study is conducted to define the frequency of Epstein-Barr virus (EBV) infection in gastric cancers of four major provinces, with different incidence rates of gastric cancers, in Iran. METHODS: Paraffin blocks of 682 cases of various types of gastric cancer from Tehran, South and North areas of Iran were collected. Twelve tissue microarray (TMA) blocks were constructed from these blocks. Localization of EBV in tumors was assessed by in situ hybridization (ISH) for EBV-encoded RNA (EBER). Chi-squared test was used to evaluate the statistical significance between EBV-associated gastric cancer (EBVaGC) and clinicopathologic tumor characteristics. RESULTS: Fourteen out of 682 cases (2.1%) of gastric adenocarcinoma were EBER-positive. EBER was positive in 8 out of 22 (36.4%) of medullary carcinomas and 6 out of 660 (0.9%) of non-medullary type, which was a statistically significant difference (P<0.001). The EBVaGCs were more frequent in younger age (P=0.009) and also showed a trend toward the lower stage of the tumor (P=0.075). CONCLUSION: EBV-associated gastric adenocarcinoma has a low prevalence in Iran. This finding can be due to epidemiologic differences in risk factors and exposures, and the low number of gastric medullary carcinomas in the population. It may also be related to gastric tumor heterogeneity not detected with the TMA technique.

In Situ Hybridization of Brain Slices.

In situ hybridization (ISH) is an important technique for identifying gene expression at the cellular level in various organs, including brain slices. This approach hybridizes nucleic acid probes to cellular mRNA, allowing the detection of transcriptional products. Recent advances have enabled RNA preservation in formalin-fixed paraffin-embedded (FFPE) samples, making ISH applicable to brain tumor diagnosis and research. Here, we provide a concise overview of the standard application of chromogenic ISH in neuroscience research and neuropathology practice using FFPE blocks of brain slice sections.

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.

RNAscope in situ hybridization reveals microvascular sequestration of Plasmodium relictum pSGS1 blood stages but absence of exo-erythrocytic dormant stages during latent infection of Serinus canaria.

BACKGROUND: Birds chronically infected with avian malaria parasites often show relapses of parasitaemia after latent stages marked by absence of parasites in the peripheral circulation. These relapses are assumed to result from the activation of dormant exo-erythrocytic stages produced during secondary (post-erythrocytic) merogony of avian Plasmodium spp. Yet, there is no morphological proof of persistent or dormant tissue stages in the avian host during latent infections. This study investigated persistence of Plasmodium relictum pSGS1 in birds with latent infections during winter, with the goal to detect presumed persisting tissue stages using a highly sensitive RNAscope® in situ hybridization technology. METHODS: Fourteen domestic canaries were infected with P. relictum pSGS1 by blood-inoculation in spring, and blood films examined during the first 4 months post infection, and during winter and spring of the following year. After parasitaemia was no longer detectable, half of the birds were dissected, and tissue samples investigated for persisting tissue stages using RNAscope ISH and histology. The remaining birds were blood-checked and dissected after re-appearance of parasitaemia, and their tissues equally examined. RESULTS: Systematic examination of tissues showed no exo-erythrocytic stages in birds exhibiting latent infections by blood-film microscopy, indicating absence of dormant tissue stages in P. relictum pSGS1-infected canaries. Instead, RNAscope ISH revealed rare P. relictum blood stages in capillaries of various tissues and organs, demonstrating persistence of the parasites in the microvasculature. Birds examined after re-appearance of parasitemia showed higher numbers of P. relictum blood stages in both capillaries and larger blood vessels, indicating replication during early spring and re-appearance in the peripheral circulation. CONCLUSIONS: The findings suggest that persistence of P. relictum pSGS1 during latent infection is mediated by continuous low-level erythrocytic merogony and possibly tissue sequestration of infected blood cells. Re-appearance of parasitaemia in spring seems to result from increased erythrocytic merogony, therefore representing recrudescence and not relapse in blood-inoculated canaries. Further, the study highlights strengths and limitations of the RNAscope ISH technology for the detection of rare parasite stages in tissues, providing directions for future research on persistence and tissue sequestration of avian malaria and related haemosporidian parasites.

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.

Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus.

Xenopus has been a powerful model organism for understanding vertebrate development and disease for over a hundred years. While experimental analysis and dissection techniques of the embryo have been well documented, descriptions of adult Xenopus structures and organs, together with techniques for working with adults, have not been updated to take into consideration the requirements of such modern approaches as quantitative proteomics and single-cell transcriptomics. The cell-type and gene-centric perspectives require contrasting observations in embryonic stages to those in adult tissues. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location all along the larval to adult transition, most notably during massive metamorphosis remodeling. Establishing robust standards for organ identification and dissection is crucial to ensure datasets resulting from studies performed at different laboratories can be consistent. The present protocol identifies six of the organs in the adult Xenopus, demonstrating methods for dissection and sampling of the heart ventricle, liver, fat body, pancreas, paired kidney, and skin of the adult Xenopus. Depending on the preservation methods, the dissected organs can be used for quantitative proteomics, single cell/nuclei transcriptomics, in situ hybridization, immunohistochemistry, histology, etc. This protocol aims to standardize tissue sampling and facilitate multi-lab investigations of the adult organ systems.

Mapping human tissues with highly multiplexed RNA in situ hybridization.

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts.

Immunohistochemical p16 overexpression and Rb loss correlate with high-risk human papillomavirus infection in endocervical adenocarcinomas.

AIMS: p16 is a sensitive surrogate marker for transcriptionally active high-risk human papillomavirus (HR-HPV) infection in endocervical adenocarcinoma (ECA); however, its specificity is not perfect. METHODS AND RESULTS: We examined p16 and Rb expressions by immunohistochemistry (IHC) and the transcriptionally active HR-HPV infection by mRNA in-situ hybridisation (ISH) with histological review in 108 ECA cases. Thirteen adenocarcinomas of endometrial or equivocal origin (six endometrioid and seven serous carcinomas) were compared as the control group. HR-HPV was detected in 83 of 108 ECA cases (77%), including five HPV-associated adenocarcinomas in situ and 78 invasive HPV-associated adenocarcinomas. All 83 HPV-positive cases showed consistent morphology, p16 positivity and partial loss pattern of Rb. Among the 25 cases of HPV-independent adenocarcinoma, four (16%) were positive for p16, and of these four cases, three of 14 (21%) were gastric type adenocarcinomas and one of 10 (10%) was a clear cell type adenocarcinoma. All 25 HPV-independent adenocarcinomas showed preserved expression of Rb irrespective of the p16 status. Similarly, all 13 cases of the control group were negative for HR-HPV with preserved expression of Rb, even though six of 13 (46%) cases were positive for p16. Compared with p16 alone, the combination of p16 overexpression and Rb partial loss pattern showed equally excellent sensitivity (each 100%) and improved specificity (100 versus 73.6%) and positive predictive values (100 versus 89.2%) in the ECA and control groups. Furthermore, HR-HPV infection correlated with better prognosis among invasive ECAs. CONCLUSIONS: The results suggest that the combined use of p16 and Rb IHC could be a reliable method to predict HR-HPV infection in primary ECAs and mimics. This finding may contribute to prognostic prediction and therapeutic strategy.

Epstein-Barr virus latency patterns in polymorphic lymphoproliferative disorders and lymphomas in immunodeficiency settings: Diagnostic implications.

Epstein-Barr virus (EBV) is responsible for many B cell lymphoproliferative disorders (LPD) spanning subclinical infection to immunodeficiency-related neoplasms. EBV establishes a latent infection in the host B cell as defined histologically by the expression of EBV latent membrane proteins and nuclear antigens. Herein, we characterize the latency patterns of immunodeficiency-related neoplasms including post-transplant lymphoproliferative disorders (PTLD) and therapy-related LPD (formerly iatrogenic) with latent membrane protein-1 (LMP-1) and EBV nuclear antigen-2 (EBNA-2) immunohistochemistry. The latency pattern was correlated with immunodeficiency and dysregulation (IDD) status and time from transplant procedure. 38 cases of EBV+ PTLD in comparison to 27 cases of classic Hodgkin lymphoma (CHL) and diffuse large B cell lymphoma (DLBCL) arising in either the therapy-related immunodeficiency setting (n = 12) or without an identified immunodeficiency (n = 15) were evaluated for EBV-encoded small RNAs by in situ hybridization (EBER-ISH) and for LMP-1 and EBNA-2 by immunohistochemistry. A full spectrum of EBV latency patterns was observed across PTLD in contrast to CHL and DLBCL arising in the therapy-related immunodeficiency setting. Polymorphic-PTLD (12 of 16 cases, 75 %) and DLBCL-PTLD (9 of 11 cases, 82 %) showed the greatest proportion of cases with latency III pattern. Whereas, EBV+ CHL in an immunocompetent patient showed exclusively latency II pattern (13 of 13 cases, 100 %). The majority of EBV+ PTLD occurred by three years of transplant procedure date and were enriched for latency III pattern (21 of 22 cases, 95 %). Immunohistochemical identification of EBV latency by LMP-1 and EBNA-2 can help classify PTLD in comparison to other EBV+ B cell LPD and lymphomas arising in therapy-related immunodeficiency and non-immunodeficiency settings.

Differential Regulation of Neurotrophic Factors During Pathogenic Tau-Aggregation in a Tau Transgenic Mouse Model for Alzheimer's Disease: A Protocol for Double-Labeling mRNA by In Situ Hybridization and Protein Epitopes by Immunohistochemistry.

Alzheimer's disease (AD), most tauopathies, and other neurodegenerative diseases are highly associated to impaired neurotrophin regulation and imbalanced neurotrophin transport and distribution. Neurotrophins are crucial for the survival and maintenance of distinct neuronal population therefore their supply is essential for a healthy brain. Tau phosphorylation occurs at different sites of the tau protein and some phospho-epitopes are highly associated to AD (e.g., abnormally phosphorylated tau at Thr212/Ser214). Though the importance of neurotrophins is well known, their analysis in tissue is not trivial and needs careful consideration. Here a detailed protocol is presented, which combines in situ hybridization (ISH) with immunohistochemistry (IHC) to analyze neurotrophin mRNA expression during tau neuropathology and the results were confirmed by immunological methods.With this protocol, it was demonstrated that Brain-Derived Neurotrophic Factor (BDNF) and its receptor Tropomyosin receptor kinase B (TrkB) were significantly decreased in tau-transgenic mice compared to their age-matched littermates. Neurotrophin-3 (NT-3) and its receptor TrkC were not altered with statistical significance, but a tendency for decreased NT-3 and slightly increased TrkC expression was observed in tau transgenic mice. The loss of BDNF-ISH signal was predominantly observed in hippocampus (CA1 and CA3) and cortex (layer II-VI) and verified by BDNF-immunoreactivity. Decreased BDNF and TrkB mRNA was negatively correlated with abnormal tau phosphorylation at Thr212/Ser214 in cortical neurons in transgenic mice. Strikingly, no correlation was observed with age-related phospho-epitopes such as Ser202/Thr205. Interestingly, both, the mRNA and protein levels of Nerve Growth Factor (NGF) were significantly increased in hippocampal neurons in the tau models as demonstrated by ISH, immunofluorescence, and Western Blotting. Here, some co-localization of NGF mRNA and phospho-tau (Thr212/Ser214) was observed but was a rare event. Since there is growing evidence for the relevance of neurotrophic factor distribution in the pathogenesis of neurodegeneration, this technique is a useful tool to investigate the underlying mechanisms and potential therapeutic intervention.

Intranasal Nose-to-Brain Drug Delivery via the Olfactory Region in Mice: Two In-Depth Protocols for Region-Specific Intranasal Application of Antibodies and for Expression Analysis of Fc Receptors via In Situ Hybridization in the Nasal Mucosa.

A region-specific catheter-based intranasal administration method was successfully developed, established, and validated as reported previously. By using this method, drugs can be applicated specifically to the olfactory region. Thereby, intranasally administered drugs could be delivered via neuronal connections to the central nervous system. Here, we present a detailed protocol with a step-by-step procedure for nose-to-brain delivery via the olfactory mucosa.Fc receptors such as the neonatal Fc receptor (FcRn) and potentially Fcγ receptor IIb (FcγRIIb) are involved in the uptake and transport of antibodies via the olfactory nasal mucosa. To better characterize their expression levels and their role in CNS drug delivery via the nose, an in situ hybridization (ISH) protocol was adapted for nasal mucosa samples and described in abundant details.

Supported Immunohistochemical Staining of Mucins.

Immunohistochemistry (IHC) staining is the most common method to detect the distribution and localization of biomarkers in different parts of a tissue. Antibodies for tandem repeat peptide of mucins are very popular, but antibodies for glycosylation or others are also used. IHC for mucin is the same protocol as IHC for others. This description includes IHC according to ABC method for processed formalin-fixed paraffin-embedded (FFPE) tissues. Protocol of in situ hybridization is also shown.

Early intestinal development of chickens divergently selected for high or low 8-wk body weight and a commercial broiler.

The early posthatch period is crucial to intestinal development, shaping long-term growth, metabolism, and health of the chick. The objective of this study was to determine the effect of genetic selection on morphological characteristics and gene expression during early intestinal development. Populations of White Plymouth Rocks have been selected for high weight (HWS) and low weight (LWS) for over 63 generations, and some LWS display symptoms of anorexia. Intestinal structure and function of these populations were compared to a commercial broiler Cobb 500 (Cobb) during the perihatch period. Egg weights, yolk-free embryo BW, yolk weights, and jejunal samples from HWS, LWS, and Cobb were collected on embryonic day (e) 17, e19, day of hatch, day (d) 3, d5, and d7 posthatch for histology and gene expression analysis. The RNAscope in-situ hybridization method was used to localize expression of the stem cell marker, olfactomedin 4 (Olfm4). Villus height (VH), crypt depth (CD), and VH/CD were measured from Olfm4 stained images using ImageJ. mRNA abundance for Olfm4, stem cell marker Lgr5, peptide transporter PepT1, goblet cell marker Muc2, marker of proliferation Ki67, and antimicrobial peptide LEAP2 were examined. Two-factor ANOVA was performed for measurements and Turkey's HSD was used for mean separation when appropriate. Cobb were heaviest and LWS the lightest (P < 0.01). at each timepoint. VH increased in Cobb and CD increased in HWS compared to LWS (P < 0.01). PepT1 mRNA was upregulated in LWS (P < 0.01), and Muc2 mRNA was decreased in both HWS and LWS compared to Cobb (P < 0.01). Selection for high or low 8-wk body weight has caused differences in intestinal gene expression and morphology when compared to a commercial broiler.

Evaluation of TRIM63 RNA in situ hybridization (RNA-ISH) as a potential biomarker for alveolar soft-part sarcoma (ASPS).

Alveolar soft-part sarcoma (ASPS) is a rare soft tissue tumor with a broad morphologic differential diagnosis. While histology and immunohistochemistry can be suggestive, diagnosis often requires exclusion of other entities followed by confirmatory molecular analysis for its characteristic ASPSCR1-TFE3 fusion. Current stain-based biomarkers (such as immunohistochemistry for cathepsin K and TFE3) show relatively high sensitivity but may lack specificity, often showing staining in multiple other entities under diagnostic consideration. Given the discovery of RNA in situ hybridization (RNA-ISH) for TRIM63 as a sensitive and specific marker of MiTF-family aberration renal cell carcinomas, we sought to evaluate its utility in the workup of ASPS. TRIM63 RNA-ISH demonstrated high levels (H-score greater than 200) of expression in 19/20 (95%) cases of ASPS (average H-score 330) and was weak or negative in cases of paraganglioma, clear cell sarcoma, rhabdomyosarcoma, malignant epithelioid hemangioendothelioma, as well as hepatocellular and adrenal cortical carcinomas. Staining was also identified in tumors with known subsets characterized by TFE3 alterations such as perivascular epithelioid cell neoplasm (PEComa, average H-score 228), while tumors known to exhibit overexpression of TFE3 protein without cytogenetic alterations, such as melanoma and granular cell tumor, generally showed less TRIM63 ISH staining (average H-scores 147 and 96, respectively). Quantitative assessment of TRIM63 staining by RNA-ISH is potentially a helpful biomarker for tumors with molecular TFE3 alterations such as ASPS.

Spatiotemporal Expression Characterization of KRTAP6 Family Genes and Its Effect on Wool Traits.

Keratin-related proteins (KAPs) are structural components of wool fibers and are thought to play a key role in regulating the physical and mechanical properties of fibers. Among all KAP genes (KRTAPs), KRTAP6 gene family (KRTAP6-1, KRTAP6-2, KRTAP6-3, KRTAP6-4, and KRTAP6-5) is a very important member with high polymorphism and notable association with some wool traits. In this study, we used real-time fluorescence quantitative PCR (RT-qPCR) and in situ hybridization to investigate spatiotemporal expression of KRTAP6s. The results revealed that KRTAP6 family genes were significantly expressed during anagen compared to other stages (p < 0.05). And it was found the five genes were expressed predominantly in the dermal papillae, inner and outer root sheaths, and showed a distinct spatiotemporal expression pattern. Also, it was found that KRTAP6-1 and KRTAP6-5 mRNA expression was negatively correlated with wool mean fiber diameter (MFD) and mean staple strength (MSS) (p < 0.05). In summary, the KRTAP6 family genes share a similar spatiotemporal expression pattern. And KRTAP6-1 and KRTAP6-5 may regulate the MFD and MSS of Gansu Alpine fine-wool sheep wool by changing the expression.

Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA.

In situ hybridization of oligonucleotide probes to intracellular RNA allows quantification of predefined gene transcripts within millions of single cells using cytometry platforms. Previous methods have been hindered by the number of RNA that can be analyzed simultaneously. Here we describe a method called proximity ligation assay for RNA (PLAYR) that permits highly multiplexed RNA analysis that can be combined with antibody staining. Potentially any number of RNA combined with antigen can be analyzed together, being limited only by the number of analytes that can be measured simultaneously.