Whole-mount MeFISH: a novel technique for simultaneous visualization of specific DNA methylation and protein/RNA expression.
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ABSTRACT:
SUBMITTER: Shiura H
PROVIDER: S-EPMC3995954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundPlant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant.ResultsWe present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode.ConclusionWe were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.
| S-EPMC10696717 | biostudies-literature
Project description:PurposeSimultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue.MethodsWhole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy.ResultsA fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue.ConclusionsThe protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.
| S-EPMC4169891 | biostudies-literature
Project description:Developing a three-dimensional (3D) visualization of the kidney at the whole-mount scale is challenging. In the present study, we optimized mouse whole-mount kidney clearing, which improved the transparency ratio to over 90% based on organ-specific perfusion (OSP)-clear, unobstructed brain imaging cocktails and computational analysis (CUBIC). The optimized OSP-CUBIC-compatible 3D immunostaining and imaging simultaneously visualized the high-resolution 3D structure of the whole-mount renal microvascular, glomerulus, and accompanying wrapped traveling sympathetic nerves in mice. A mouse model of pressure overload-induced heart failure (HF) was then established by minimally invasive transverse aortic constriction (MTAC). Further 3D quantification revealed renal sympathetic hyperinnervation (6.80 ± 1.04% vs. 3.73 ± 0.60%, P < 0.05) in mice with HF. In conclusion, this newly developed whole-organ tissue clearing and imaging system provides comprehensive information at the whole-mount scale and has great potential for kidney research. Our data suggest that renal sympathetic hyperinnervation is involved in HF associated with renal dysfunction.
| S-EPMC8297621 | biostudies-literature
Project description:Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described. Finally, a suite of custom-written ImageJ macros is included with detailed instructions to enable semi-automated smFISH image analysis of both 2D and 3D images. © 2018 by John Wiley & Sons, Inc.
| S-EPMC6500779 | biostudies-literature
Project description:Aberrant DNA methylation plays an important role in cancer progression. However, no resource has been available that comprehensively provides DNA methylation-based diagnostic and prognostic models, expression-methylation quantitative trait loci (emQTL), pathway activity-methylation quantitative trait loci (pathway-meQTL), differentially variable and differentially methylated CpGs, and survival analysis, as well as functional epigenetic modules for different cancers. These provide valuable information for researchers to explore DNA methylation profiles from different aspects in cancer. To this end, we constructed a user-friendly database named DNA Methylation Interactive Visualization Database (DNMIVD), which comprehensively provides the following important resources: (i) diagnostic and prognostic models based on DNA methylation for multiple cancer types of The Cancer Genome Atlas (TCGA); (ii) meQTL, emQTL and pathway-meQTL for diverse cancers; (iii) Functional Epigenetic Modules (FEM) constructed from Protein-Protein Interactions (PPI) and Co-Occurrence and Mutual Exclusive (COME) network by integrating DNA methylation and gene expression data of TCGA cancers; (iv) differentially variable and differentially methylated CpGs and differentially methylated genes as well as related enhancer information; (v) correlations between methylation of gene promoter and corresponding gene expression and (vi) patient survival-associated CpGs and genes with different endpoints. DNMIVD is freely available at http://www.unimd.org/dnmivd/. We believe that DNMIVD can facilitate research of diverse cancers.
| S-EPMC6943050 | biostudies-literature
Project description:BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.
| S-EPMC4172952 | biostudies-literature
Project description:A novel mutagenesis technique using error-prone DNA polymerase ? (pol?), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the pol?MKII mutator, which expresses the proofreading-deficient and low-fidelity pol?, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS). This mutator strain possesses exogenous mutant pol? supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the pol?MKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.
| S-EPMC3364565 | biostudies-literature
Project description:ABSTRACT The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms. Summary: A new deyolking method for zebrafish embryos enables high-resolution confocal imaging of deep tissues, offering a simple and effective technique for biomedical research.
| S-EPMC10373579 | biostudies-literature
Project description:Although a high-resolution three-dimensional mapping system has made it possible to treat complicated atrial tachyarrhythmia (AT), there remain cases that are difficult to diagnose and treat. However, when multiple different ATs alternately appear, mapping and diagnosis of those are more difficult. Parallel mapping module is well known as a good option to simultaneously map two or more different ATs when they alternately appear. When performing parallel mapping of two different ATs, one bipolar signal of the reference catheter is used as a timing reference and a cycle length filter is used for differentiating AT1, AT2, and others, including sinus rhythm, fusion beats, or catheter-induced premature atrial complex. Therefore, it has some limitations for differentiating multifocal ATs. We present a case wherein multifocal ATs were successfully eliminated by combining parallel mapping module and dual-chamber intra-cardiac pattern matching technique that we have previously reported.Learning objective▪Parallel mapping is a useful tool when two or more tachycardias alternately occur; however, it has some limitations.▪Dual-chamber intra-cardiac pattern matching technique, which combines right atrial and coronary sinus potentials, provides better discrimination than coronary sinus reference alone.▪By combining parallel mapping and dual-chamber intra-cardiac pattern matching, two or more atrial tachycardias could be automatically and simultaneously mapped.
| S-EPMC10865130 | biostudies-literature
Project description:DNA methylation plays critical roles in gene regulation and cellular specification without altering DNA sequences. The wide application of reduced representation bisulfite sequencing (RRBS) and whole genome bisulfite sequencing (bis-seq) opens the door to study DNA methylation at single CpG site resolution. One challenging question is how best to test for significant methylation differences between groups of biological samples in order to minimize false positive findings.We present a statistical analysis package, methylSig, to analyse genome-wide methylation differences between samples from different treatments or disease groups. MethylSig takes into account both read coverage and biological variation by utilizing a beta-binomial approach across biological samples for a CpG site or region, and identifies relevant differences in CpG methylation. It can also incorporate local information to improve group methylation level and/or variance estimation for experiments with small sample size. A permutation study based on data from enhanced RRBS samples shows that methylSig maintains a well-calibrated type-I error when the number of samples is three or more per group. Our simulations show that methylSig has higher sensitivity compared with several alternative methods. The use of methylSig is illustrated with a comparison of different subtypes of acute leukemia and normal bone marrow samples.methylSig is available as an R package at http://sartorlab.ccmb.med.umich.edu/software.Supplementary data are available at Bioinformatics online.
| S-EPMC4147891 | biostudies-other