In situ hybridization is a commonly used technique in molecular biology to assess the temporal and spatial expression of a given gene. As a long and labor-intensive protocol, double in situ hybridization, which detects two genes in series, is challenging and can require a lot of troubleshooting. Optional additives, polyvinyl alcohol and dextran sulfate, were tested in a standard in situ hybridization protocol and several colorimetric stain pairings using double in situ hybridization in zebrafish embryos. Optional additives can improve staining time and reduce nonspecific background. Nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (BCIP) + Fast Red/BCIP was the most effective stain pairing. As a proof-of-concept, this work shows that Cabin1 and atoh1b are expressed in distinct regions of the developing zebrafish brain.

In situ hybridization is a standard procedure for visualizing mRNA transcripts in tissues. The recent adoption of fluorescent probes and new signal amplification methods have facilitated multiplexed RNA imaging in tissue sections and whole tissues. Here we present protocols for multiplexed hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) staining, imaging, cell segmentation, and mRNA quantification in regenerating axolotl tissue sections. We also present a protocol for whole-mount staining and imaging of developing axolotl limbs.

Separation of cell types and developmental stages in the Arabidopsis root and subsequent expression profiling have yielded a valuable dataset that can be used to select candidate genes for detailed study and to start probing the complexities of gene regulation in plant development.

Valid determination of HER2 status is a prerequisite to establish an adequate treatment strategy for breast cancer patients, regardless of the disease stage. The goal of this study was to examine the feasibility of the newly developed silver-enhanced in situ hybridization (SISH) technique as an alternative to fluorescence in situ hybridization (FISH) for HER2 assay in primary invasive breast cancer.

The objective of this study was to develop digoxigenin-labeled in situ hybridization (ISH) for the detection of Streptococcus suis in naturally infected pigs with polyserositis and to compare it with biotinylated ISH. Digoxigenin-labeled hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis and microcolonies in the blood vessels. Mock hybridization showed no hybridization signals for endogenous digoxigenin. Biotinylated hybridization signals for S. suis were observed in cells that had infiltrated the fibrous polyserositis. However, similar hybridization signals were also observed in the fibrous inflammatory area using mock hybridization for endogenous biotin. The present study demonstrated that digoxigenin-labeled ISH is a valuable diagnostic tool for specific detection of S. suis in polyserositic tissues without nonspecific reactions compared with biotinylated ISH.

This paper describes a micro fluorescence in situ hybridization (μFISH)-based rapid detection of cytogenetic biomarkers on formalin-fixed paraffin embedded (FFPE) tissue sections. We demonstrated this method in the context of detecting human epidermal growth factor 2 (HER2) in breast tissue sections. This method uses a non-contact microfluidic scanning probe (MFP), which localizes FISH probes at the micrometer length-scale to selected cells of the tissue section. The scanning ability of the MFP allows for a versatile implementation of FISH on tissue sections. We demonstrated the use of oligonucleotide FISH probes in ethylene carbonate-based buffer enabling rapid hybridization within <1 min for chromosome enumeration and 10-15 min for assessment of the HER2 status in FFPE sections. We further demonstrated recycling of FISH probes for multiple sequential tests using a defined volume of probes by forming hierarchical hydrodynamic flow confinements. This microscale method is compatible with the standard FISH protocols and with the Instant Quality FISH assay and reduces the FISH probe consumption ∼100-fold and the hybridization time 4-fold, resulting in an assay turnaround time of <3 h. We believe that rapid μFISH has the potential of being used in pathology workflows as a standalone method or in combination with other molecular methods for diagnostic and prognostic analysis of FFPE sections.

Circulating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.

Single molecule RNA imaging using fluorescent in situ hybridization (FISH) can provide quantitative information on mRNA abundance and localization in a single cell. There is now a growing interest in screening for modifiers of RNA abundance and/or localization. For instance, microsatellite expansion within RNA can lead to toxic gain-of-function via mislocalization of these transcripts into RNA aggregate and sequestration of RNA-binding proteins. Screening for inhibitors of these RNA aggregate can be performed by high-throughput RNA FISH. Here we describe detailed methods to perform single molecule RNA FISH in multiwell plates for high-content screening (HCS) microscopy. We include protocols adapted for HCS with either standard RNA FISH with fluorescent oligonucleotide probes or the recent single molecule inexpensive FISH (smiFISH). Recommendations for success in HCS microscopy with high magnification objectives are discussed.

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing approximately 1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades.

Marker chromosomes are small supernumerary chromosomes that cannot be unambiguously identified by chromosome banding techniques alone. However, the precise characterization of marker chromosomes is important for prenatal diagnosis and proper genetic counseling. In this study, we evaluated the chromosomal origin of marker chromosomes using a combination of banding cytogenetics and molecular cytogenetic techniques including diverse fluorescence in situ hybridization (FISH) assays and array comparative genomic hybridization (array CGH).

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

The visualization of multiple gene expressions in well-preserved tissues is crucial for the elucidation of physiological and pathological processes. In situ hybridization chain reaction (HCR) is a method to visualize specific mRNAs in diverse organisms by applying a HCR that is an isothermal enzyme-free nucleotide polymerization method using hairpin DNAs. Although in situ HCR is a versatile method, this method is not widely used by researchers because of their higher cost than conventional in situ hybridization (ISH). Here, we redesigned hairpin DNAs so that their lengths were half the length of commonly used hairpin DNAs. We also optimized the conjugated fluorophores and linkers. Modified in situ HCR showed sufficient fluorescent signals to detect various mRNAs such as Penk, Oxtr, Vglut2, Drd1, Drd2, and Moxd1 in mouse neural tissues with a high signal-to-noise ratio. The sensitivity of modified in situ HCR in detecting the Oxtr mRNA was better than that of fluorescent ISH using tyramide signal amplification. Notably, the modified in situ HCR does not require proteinase K treatment so that it enables the preservation of morphological structures and antigenicity. The modified in situ HCR simultaneously detected the distributions of c-Fos immunoreactivity and Vglut2 mRNA, and detected multiple mRNAs with a high signal-noise ratio at subcellular resolution in mouse brains. These results suggest that the modified in situ HCR using short hairpin DNAs is cost-effective and useful for the visualization of multiple mRNAs and proteins.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) represent a global challenge in public health. It is estimated that 50 to 100 million infections occur each year causing approximately 20,000 deaths that are usually linked to severe cases like DHF and dengue shock syndrome. The causative agent of DF is dengue virus (genus Flavivirus) that comprises four distinct serotypes (DENV-1 to DENV-4). Fluorescence in situ hybridization (FISH) has been used successfully to detect pathogenic agents, but has not been implemented in detecting DENV. To improve our understanding of DENV infection and dissemination in host tissues, we designed specific probes to detect DENV in FISH assays.

Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

Apicomplexan parasites of the genus Babesia cause babesiosis in humans and animals. The microscopic examination of stained blood smears, detection of serum antibodies by immunoassays, and PCR-based identification of parasite nucleic acid in blood are common laboratory methods for diagnosing babesiosis. The present study evaluated a commercially available Babesia genus-specific fluorescence in situ hybridization (FISH) test for detecting Babesia parasites in blood smears. The FISH test detected Babesia duncani and Babesia microti, two common species that cause human infections in the USA, and other Babesia species of human and veterinary importance in less than two hours. The Babesia genus-specific FISH test supplements other existing laboratory methods for diagnosing babesiosis and may be particularly useful in resource-limited laboratories.

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. Recently there has been a surge of multiplexed RNA in situ techniques but their application to human tissues and clinical biopsies has been limited due to their large size, general lower tissue quality and high background autofluorescence. Here we report DART-FISH, a versatile padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections at cellular resolution. We introduced an omni-cell type cytoplasmic stain, dubbed RiboSoma that substantially improves the segmentation of cell bodies. We developed a computational decoding-by-deconvolution workflow to extract gene spots even in the presence of optical crowding. Our enzyme-free isothermal decoding procedure allowed us to image 121 genes in a large section from the human neocortex in less than 10 hours, where we successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. Additionally, we demonstrated the detection of transcripts as short as 461 nucleotides, including neuropeptides and discovered new cortical layer markers. 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.

MicroRNAs (miRNAs) play key roles in gene expression regulation in both healthy and disease brains. To better understand those roles, it is necessary to characterize the miRNAs that are expressed in particular cell types under a range of conditions. In situ hybridization (ISH) can demonstrate cell- and lamina-specific patterns of miRNA expression that would be lost in tissue-level expression profiling. In the present study, ISH was performed with special focus on the human entorhinal cortex (EC) and transentorhinal cortex (TEC). The TEC is the area of the cerebral cortex that first develops neurofibrillary tangles in Alzheimer's disease (AD). However, the reason for TEC's special vulnerability to AD-type pathology is unknown. MiRNA ISH was performed on three human brains with well-characterized clinical and pathological parameters. Locked nucleic acid ISH probes were used referent to miR-107, miR-124, miR-125b, and miR-320. In order to correlate the ISH data with AD pathology, the ISH staining was compared with near-adjacent slides processed using Thioflavine stains. Not all neurons or cortical lamina stain with equal intensity for individual miRNAs. As with other areas of brain, the TEC and EC have characteristic miRNA expression patterns. MiRNA ISH is among the first methods to show special staining characteristics of cells and laminae of the human TEC.

MicroRNAs (miRNAs) participate in a variety of functions in the brain. Understanding the in vivo localization of miRNAs is an important step for uncovering their roles in brain function. However, the in situ detection of low-abundance miRNAs in brain tissues remains difficult and requires extensive optimization of in situ hybridization (ISH) protocols in individual laboratories. Thus, detailed information regarding experimental conditions would serve as a useful reference for researchers in this field. Here, we investigated and summarized the effects of adjusting a series of critical steps, including tissue fixation, probe accessibility and hybridization stringency, to standardize the currently used miRNA ISH procedures. As a result, we successfully detected several low-abundance miRNAs by ISH using the following experimental conditions: (1) use of fresh brain tissues, (2) digestion of brain samples with proteinase K, (3) LNA-probe hybridization at a temperature 37°C below the melting temperature of the RNA, (4) performance of high-stringency wash steps using 50% formamide in 1 × standard saline citrate (SSC) buffer. RT-PCR of the punched-out tissues using TaqManTM primers confirmed the ISH results. Finally, double-fluorescence ISH successfully demonstrated the colocalization of miRNAs and mRNAs. Thus, the detailed information regarding the miRNA ISH procedures used in this study may help to resolve the technical hurdles observed in the in vivo localization of miRNAs, and the elucidation of the specific roles of miRNAs.