WISH-BONE: Whole-mount in situ histology, to label osteocyte mRNA and protein in 3D adult mouse bones.

Bone is a three-dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two-dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte messenger RNA (mRNA) and protein in whole-mount mouse bone. For mRNA labeling, we used hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.

Use of FISH-FLOW as a Method for the Identification and Quantification of Bacterial Populations.

The gastrointestinal tract (GIT) harbors the largest group of microbiotas among the microbial communities of the human host. The resident organisms typical of a healthy gut are well adapted to the gastrointestinal environment while alteration of these populations can trigger disorders that may affect the health and well-being of the host. Various investigations have applied different tools to study bacterial communities in the gut and their correlation with gastrointestinal disorders such as inflammatory bowel disease (IBD), obesity, and diabetes. This study proposes fluorescent in situ hybridization, combined with flow cytometry (FISH-FLOW), as an alternative approach for phylum level identification of Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria and quantification of target bacteria from the GIT based on analysis of fecal samples, where results are validated by quantitative polymerase chain reaction (qPCR) and 16S ribosomal ribonucleic acid (16s rRNA) sequencing. The results obtained via FISH-FLOW experimental approach show high specificity for the developed probes for hybridization with the target bacteria. The study, therefore, suggests the FISH-FLOW as a reliable method for studying bacterial communities in the gut with results correlating well with those of metagenomic investigations of the same fecal samples.

Review of In Situ Hybridization (ISH) Stain Images Using Computational Techniques.

Recent advancements in medical imaging have greatly enhanced the application of computational techniques in digital pathology, particularly for the classification of breast cancer using in situ hybridization (ISH) imaging. HER2 amplification, a key prognostic marker in 20-25% of breast cancers, can be assessed through alterations in gene copy number or protein expression. However, challenges persist due to the heterogeneity of nuclear regions and complexities in cancer biomarker detection. This review examines semi-automated and fully automated computational methods for analyzing ISH images with a focus on HER2 gene amplification. Literature from 1997 to 2023 is analyzed, emphasizing silver-enhanced in situ hybridization (SISH) and its integration with image processing and machine learning techniques. Both conventional machine learning approaches and recent advances in deep learning are compared. The review reveals that automated ISH analysis in combination with bright-field microscopy provides a cost-effective and scalable solution for routine pathology. The integration of deep learning techniques shows promise in improving accuracy over conventional methods, although there are limitations related to data variability and computational demands. Automated ISH analysis can reduce manual labor and increase diagnostic accuracy. Future research should focus on refining these computational methods, particularly in handling the complex nature of HER2 status evaluation, and integrate best practices to further enhance clinical adoption of these techniques.

Mixed Gonadal Dysgenesis with 45,X/46,X,idic(Y)/46,XY Karyotype: A Case Report.

Background: The purpose of the current study was to report a case with 45,X/46,XY/46,X,idic(Yp) mosaicism showing the male phenotype with mixed gonadal dysgenesis. Case Presentation: A 27 year-old individual, phenotypically male, presented with azoospermia and a micropenis. Both testes were not visualized in the scrotal sac. Due to the presence of a small-sized uterus, the individual was referred to the KSHEMA Center for Genetic Services for chromosomal analysis. Karyotyping revealed a mosaic karyotype of 45,X[44]/46,XY[5]/46,X,idic(Yp)[1]. This finding was further confirmed through fluorescent in situ hybridization (FISH) analysis. The individual's mosaic karyotype consisted of three cell lines, with a higher proportion of the 45,X cell line and lower proportions of the idic(Yp) and 46,XY cell lines. It is worth noting that this mosaic condition in postnatal peripheral blood has not been reported in the literature thus far. Conclusion: The case report demonstrated the importance of performing karyotype and FISH analysis in understanding genetic defects including mosaicism and other chromosomal aberrations, which can influence not only growth and puberty but also sexual development and maturation. Hence, performing cytogenetic and molecular cytogenetic analysis will help clinicians to take a further step in understanding and managing the condition.

In silico Characterization of Satellitomes and Cross-Amplification of Putative satDNAs in Two Species of the Hypostomus ancistroides Complex (Siluriformes, Loricariidae).

INTRODUCTION: The mapping of the satellite DNA on chromosomes is vital to understanding the distribution and evolution of repetitions in the genome since these chromosomal studies have shown the origin, evolutionary mode, and function of repetitive sequences. This study aimed to prospect the satellitome and determine its location in the genome of two cryptic species of Hypostomus, H. aff. ancistroides and H. ancistroides, with and without XX/XY sexual chromosome system. METHODS: Mitotic chromosomes and DNA extraction were obtained according to protocols. After the whole genome sequencing, the satDNAs were retrieved, amplified, and hybridized in chromosome preparations for male and female individuals. RESULTS: We found 30 satellite families (47 variants, two superfamilies) in H. ancistroides and 38 satellite families (45 variants, four superfamilies) in H. aff. ancistroides. The sequences varied from 14 bp to 2,662 bp in H. ancistroides and from 14 bp to 2,918 bp in H. aff. ancistroides. We did not observe any tandem repeats that were exclusive to each of the libraries; however, many sequences showed very different abundances and copy numbers between the libraries. Four satDNAs did not hybridize on the chromosomes of either species. Conversely, one satDNA hybridized in both species, HxySat1-80. However, the phenotypes found varied among species, populations, and in the same individual. There was no sign of HanSat3-464 and HanSat11-335 in any individuals of H. aff. ancistroides, but markings were in the chromosomes of H. ancistroides. HxySat12-1127 and HxySat8-52, on the other hand, were only hybridized in H. aff. ancistroides, while H. ancistroides had a negative sign. No hybridization of satDNAs was found in the X and Y sex chromosomes as they were mostly composed of euchromatin. CONCLUSION: We distinguish H. aff. ancistroides as genetically different from H. ancistroides, recognizing that such characteristics go far beyond morphological, karyotypic, and molecular data. Our data support the differential abundance and location of satellite DNAs and confirm that many organisms, including fish, have repetitive sequences that validate the library hypothesis. All found and validated satDNAs and the characterization of the satellitomes of the two species represent important contributions to cytogenomic studies of the genus Hypostomus.

Shifting from Immunohistochemistry to Screen for ALK Rearrangements: Real-World Experience in a Large Single-Center Cohort of Patients with Non-Small-Cell Lung Cancer.

The identification of ALK fusions in advanced non-small-cell lung carcinoma (aNSCLC) is mandatory for targeted therapy. The current diagnostic approach employs an algorithm using ALK immunohistochemistry (IHC) screening, followed by confirmation through ALK FISH and/or next-generation sequencing (NGS). Challenges arise due to the infrequency of ALK fusions (3-7% of aNSCLC), the suboptimal specificity of ALK IHC and ALK FISH, and the growing molecular demands placed on small tissue samples, leading to interpretative, tissue availability, and time-related issues. This study investigates the effectiveness of RNA NGS as a reflex test for identifying ALK fusions in NSCLC, with the goal of replacing ALK IHC in the systematic screening process. The evaluation included 1246 NSCLC cases using paired techniques: ALK IHC, ALK FISH, and ALK NGS. ALK IHC identified 51 positive cases (4%), while RNA NGS detected ALK alterations in 59 cases (4.8%). Of the 59 ALK-positive cases identified via NGS, 53 (89.8%) were confirmed to be positive. This included 51 cases detected via both FISH and IHC, and 2 cases detected only via FISH, as they were completely negative according to IHC. The combined reporting time for ALK IHC and ALK FISH averaged 13 days, whereas ALK IHC and RNA NGS reports were obtained in an average of 4 days. These results emphasize the advantage of replacing systematic ALK IHC screening with RNA NGS reflex testing for a more comprehensive and accurate assessment of ALK status.

Carica papaya L. sex chromosome review and physical mapping of the serk 2, svp-like and mdar 4 sequences.

Physical mapping evidences the chromosome organization and structure. Despite the data about plant cytogenomics, physical mapping has been conducted from single-copy and/or low-copy genes for few species. Carica papaya cytogenomics has been accomplished from BAC-FISH and repeatome sequences. We aimed to map the serk 2, svp-like and mdar 4 sequences in C. papaya. The sequences were amplified and the amplicons sequenced, showing similarity in relation to serk 2, svp-like and mdar 4 genes. Carica papaya diploidy was confirmed and the mitotic chromosomes characterized. The chromosome 1 exhibited the secondary constriction pericentromeric to the centromere of the long arm. So, we concluded that it is the sex chromosomes. serk 2 was mapped in the long arm interstitial portion of the sex chromosomes, and the interphase nuclei showed two fluorescence signals. Considering these results and the sequencing data from the C. papaya sex chromosomes, svp-like and mdar 4 genes were mapped in the interstitial region of the sex chromosome long arm. Both sequences showed only one fluorescence signal in the interphase nuclei. The procedure adopted here can be reproduced for other single-copy and/or low-copy genes, allowing the construction of cytogenetic maps. In addition, we revisited the cytogenomics data about C. papaya sex chromosomes, presenting a revised point of view about the structure and evolution to these chromosomes.

An alternative method for SARS-CoV-2 detection with use modified fluorescent in situ hybridization.

The real-time reverse-transcriptase polymerase-chain-reaction (rRT-PCR) tests are the gold standard in detecting SARS-CoV-2 virus infection. However, despite high sensitivity and specificity, they have limitations that in some cases may result in false negative results. Therefore, it is reasonable to search for additional tools that could support microbiological diagnosis of SARS-CoV-2. The aim of the study was to develop a highly specific molecular test capable of detecting and visualizing SARS-CoV-2 infection. A universal probe and a set of 18 specific oligonucleotides with a FLAP sequence attached to them on both sides were designed to visualize SARS-CoV-2 virus infection based on the fluorescence in situ hybridization method (FISH). FISH conditions using the developed kit were standardized on the Vero CCL-81 cell line infected by SARS-CoV-2 virus. The method was tested on 290 nasopharyngeal swabs (collected in a doublet) from patients with clinical symptoms of SARS-CoV-2. Each one swab from the doublet was subjected to RNA isolation and amplification by rRT-PCR. From the second swab, a microscopic preparation was performed for FISH. The use of the rRT-PCR allowed obtaining 200 positive and 90 negative results, while our FISH method allowed for 220 positive results and 70 negative results. The differences obtained using both methods were statistically significant (p = 0.008). The obtained results support the use of FISH as an additional method in microbiological diagnostics of SARS-CoV-2.

Spatial recovery of the murine gut microbiota after antibiotics perturbation.

Bacterial communities are highly complex, with interaction networks dictating ecosystem function. Bacterial interactions are constrained by the spatial organization of these microbial communities, yet studying the spatial organization of microbial communities at the single-cell level has been technically challenging. Here, we use the recently developed high-phylogenetic-resolution microbiota mapping by fluorescence in situ hybridization technology to image the gut microbiota at the species and single-cell level. We simultaneously image 63 different bacterial species to spatially characterize the perturbation and recovery of the gut microbiota to ampicillin and vancomycin in the cecum and distal colon of mice. To decipher the biology in this complex imaging data, we developed an analytical framework to characterize the spatial changes of the gut microbiota to a perturbation. The three-tiered analytical approach includes image-level diversity, pairwise colocalization analysis, and hypothesis-driven neighborhood analysis. Through this workflow, we identify biogeographic and antibiotic-based differences in the spatial organization of the gut microbiota. We demonstrate that the cecal microbiota has increased micrometer-scale diversity than the colon at baseline and recovers better from perturbation. Also, we identify potential foundation and keystone species that have high baseline neighborhood richness and that are associated with recovery from antibiotics. Through this workflow, we add a spatial layer to the characterization of bacterial communities and progress toward a better understanding of bacterial interactions leading to improved microbiome modulation strategies. IMPORTANCE: Antibiotics have broad off-target effects on the gut microbiome. When the microbial community is unable to recover from antibiotics, it can lead to increased susceptibility to gastrointestinal infections and increased risk of immunological and metabolic diseases. In this study, we work to better understand how the gut microbiota recovers from antibiotics by employing a recent technology to image the entire bacterial community at once. Through this approach, we characterize the spatial changes in the gut microbiota after treatment with model antibiotics in both the cecum and colon of mice. We find antibiotic- and biogeographic-dependent spatial changes between bacterial species and that many of these spatial colocalizations do not recover to baseline levels even 35 days after antibiotic administration.

Clinical and pathological characteristics of renal cell carcinomas with MiTF translocation.

INTRODUCTION: Microphthalmia Transfactor Family (MiTF) translocation renal cell carcinomas (RCCs) represent a rare subtype of renal cell cancers. They are diagnosed in young patients and have a poor prognosis. The aim of our study was to analyze the clinical and pathological features of patients with MiTF RCC. MATERIAL AND METHOD: We performed a retrospective, monocentric, descriptive study including all patients operated for RCC between January 2015 and January 2023. The diagnosis of MiTF RCC was suspected by immunohistochemistry (IHC) and confirmed by fluorescent in situ hybridization (FISH). Survival data according to histological subtype (MiTF versus ccRCC) were analyzed using the Kaplan-Meier method and compared using a log-rank test. The primary endpoint was recurrence-free survival (RFS). A descriptive cohort analysis was performed. RESULTS: Of the 960 patients included, 19 (2%) had FISH-confirmed MiTF tumors. The median age at diagnosis was 42 years [18-75], the sex ratio was 1.11 females for 1 male, and 4 (21%) patients were immediately metastatic. Median RFS was 21months for patients in the MiTF group and was significantly lower than that of ccRCC patients, HR=4.33 [CI95% 2.06; 9.10; P<0.001]. Of the 11 patients with cT1-T2 tumors, 9 (81.8%) were treated with nephron sparing-surgery, with 2 (22.2%) harbored local recurrence. CONCLUSION: Our study shows that patients with MiTF translocation RCC have a significantly lower RFS than non-MiTF RCC patients. Nephron sparing surgery must be weighted by the high risk of recurrence in this particularly young population.

Combining Double-Dose and High-Dose Pulsed Dapsone Combination Therapy for Chronic Lyme Disease/Post-Treatment Lyme Disease Syndrome and Co-Infections, Including Bartonella: A Report of 3 Cases and a Literature Review.

Three patients with relapsing and remitting borreliosis, babesiosis, and bartonellosis, despite extended anti-infective therapy, were prescribed double-dose dapsone combination therapy (DDDCT) for 8 weeks, followed by one or several two-week courses of pulsed high-dose dapsone combination therapy (HDDCT). We discuss these patients' cases to illustrate three important variables required for long-term remission. First, diagnosing and treating active co-infections, including Babesia and Bartonella were important. Babesia required rotations of multiple anti-malarial drug combinations and herbal therapies, and Bartonella required one or several 6-day HDDCT pulses to achieve clinical remission. Second, all prior oral, intramuscular (IM), and/or intravenous (IV) antibiotics used for chronic Lyme disease (CLD)/post-treatment Lyme disease syndrome (PTLDS), irrespective of the length of administration, were inferior in efficacy to short-term pulsed biofilm/persister drug combination therapy i.e., dapsone, rifampin, methylene blue, and pyrazinamide, which improved resistant fatigue, pain, headaches, insomnia, and neuropsychiatric symptoms. Lastly, addressing multiple factors on the 16-point multiple systemic infectious disease syndrome (MSIDS) model was important in achieving remission. In conclusion, DDDCT with one or several 6-7-day pulses of HDDCT, while addressing abnormalities on the 16-point MSIDS map, could represent a novel effective clinical and anti-infective strategy in CLD/PTLDS and associated co-infections including Bartonella.

Different Nuclear Architecture in Human Sperm According to Their Morphology.

Human sperm parameters serve as a first step in diagnosing male infertility, but not in determining the potential for successful pregnancy during assisted reproductive technologies (ARTs) procedures. Here, we investigated the relationship between sperm head morphology at high magnification, based on strict morphologic criteria, and the nuclear architecture analyzed by fluorescence in situ hybridization (FISH). We included five men. Two of them had an elevated high-magnification morphology score of 6 points (Score 6) indicating high fertility potential, whereas three had a low score of 0 points (Score 0), indicating low fertility potential. We used FISH to study the inter-telomeric distance and the chromosomal territory area of chromosome 1 (Chr. 1). We then compared these two parameters between subjects with high and low scores. FISH data analysis showed that the inter-telomeric distance (ITD) and chromosomal territory area (CTA) of Chr. 1 were significantly higher in subjects with low scores (score 0) than high scores (score 6). Our results suggest that (i) there is a link between nuclear architecture and sperm head abnormalities, particularly vacuoles; and (ii) it is possible to select spermatozoa with normal nuclear architecture, which might indirectly explain the positive ART outcomes observed with this technique.

The cell-type-specific spatial organization of the anterior thalamic nuclei of the mouse brain.

Understanding the cell-type composition and spatial organization of brain regions is crucial for interpreting brain computation and function. In the thalamus, the anterior thalamic nuclei (ATN) are involved in a wide variety of functions, yet the cell-type composition of the ATN remains unmapped at a single-cell and spatial resolution. Combining single-cell RNA sequencing, spatial transcriptomics, and multiplexed fluorescent in situ hybridization, we identify three discrete excitatory cell-type clusters that correspond to the known nuclei of the ATN and uncover marker genes, molecular pathways, and putative functions of these cell types. We further illustrate graded spatial variation along the dorsomedial-ventrolateral axis for all individual nuclei of the ATN and additionally demonstrate that the anteroventral nucleus exhibits spatially covarying protein products and long-range inputs. Collectively, our study reveals discrete and continuous cell-type organizational principles of the ATN, which will help to guide and interpret experiments on ATN computation and function.

Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH.

N6-methyladenosine (m6A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m6A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.

Hi-M: A Multiplex Oligopaint FISH Method to Capture Chromatin Conformations In Situ and Accompanying Open-Source Acquisition Software.

The simultaneous observation of three-dimensional (3D) chromatin structure and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or is affected by other processes, such as transcription. We have recently introduced an innovative technology known as Hi-M, which enables the sequential tagging, 3D visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this chapter, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete Hi-M acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.

hcHCR: High-Throughput Single-Cell Imaging of RNA in Human Primary Immune Cells.

Functional genomics and chemical screens can identify and characterize novel cellular factors regulating signaling networks and chemical tools to modulate their function for the treatment of disease. Screening methods have relied primarily on immortalized and/or transformed cancer cell lines, which can limit the generalization of results to more physiologically relevant systems. Most have also relied on immunofluorescence, or on stably expressed recombinant fluorescent proteins, to detect specific protein markers using high-content imaging readouts. In comparison, high-throughput methods to visualize and measure RNA species have been less explored. To address this, we have adapted an isothermal signal amplification chemistry for RNA FISH known as hybridization chain reaction (HCR) to an automated, high-content imaging assay format. We present a detailed protocol for this technique, which we have named high-content HCR (hcHCR). The protocol focuses on the measurement of changes in mRNA abundance at the single-cell level in human primary cells, but it can be applied to a variety of primary cell types and perturbing agents. We anticipate that hcHCR will be most suitable for low- to medium-throughput screening experiments in which changes in transcript abundance are the desired output measure.

Spaghetti Connections: Synaptonemal Complexes as a Tool to Explore Chromosome Structure, Evolution, and Meiotic Behavior in Fish.

BACKGROUND: The synaptonemal complex (SC) is a protein axis formed along chromosomes during meiotic prophase to ensure proper pairing and crossing over. SC analysis has been widely used to study the chromosomes of mammals and less frequently of birds, reptiles, and fish. It is a promising method to investigate the evolution of fish genomes and chromosomes as a part of complex approach. SUMMARY: Compared with conventional metaphase chromosomes, pachytene chromosomes are less condensed and exhibit pairing between homologous chromosomes. These features of SCs facilitate the study of the small chromosomes that are typical in fish. Moreover, it allows the study of heteromorphisms in sex chromosomes and supernumerary chromosomes. In addition, it enables the investigation of the pairing between orthologous chromosomes in hybrids, which is crucial for uncovering the causes of hybrid sterility and asexual reproduction, such as gynogenesis or hybridogenesis. However, the application of SC analysis to fish chromosomes is limited by the associated complications. First, in most fish, meiosis does not occur during every season and life stage. Second, different SC preparation methods are optimal for different fish species. Third, commercial antibodies targeting meiotic proteins have been primarily developed against mammalian antigens, and not all of them are suitable for fish chromosomes. KEY MESSAGES: In the present review, we provide an overview of the methods for preparing fish SCs and highlight important studies using SC analysis in fish. This study will be valuable for planning and designing research that applies SC analysis to fish cytogenetics and genomics.

Measurable residual disease study through three different methods can anticipate relapse and guide early interventions in childhood acute lymphoblastic leukemia

Introduction Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Measurable residual disease (MRD, previously named minimal residual disease) study can guide therapy adjustments or preemptive interventions that might avoid hematological relapse. Methods Clinical decision making and patient outcome were evaluated in 80 real-life childhood ALL patients, according to the results observed in 544 bone marrow samples analyzed with three MRD methods: multiparametric flow cytometry (MFC), fluorescent in-situ hybridization (FISH) on B or T-purified lymphocytes and patient-specific nested reverse transcription polymerase chain reaction (RT-PCR). Results Estimated 5 year overall survival and event-free survival were 94% and 84.1%, respectively. A total of 12 relapses in 7 patients were associated with positive MRD detection with at least one of the three methods: MFC (p < 0.00001), FISH (p < 0.00001) and RT-PCR (p = 0.013). MRD assessment allowed the anticipation of relapse and adapted early interventions with different approaches including chemotherapy intensification, blinatumomab, HSCT and targeted therapy to halt relapse in five patients, although two of them relapsed afterwards. Conclusion MFC, FISH and RT-PCR are complementary methods for MRD monitoring in pediatric ALL. Although, our data clearly show that MDR positive detection is associated with relapse, continuation of standard treatment, intensification or other early interventions were able to halt relapse in patients with different risks and genetic background. More sensitive and specific methods are warranted to enhance this approach. However, whether early treatment of MRD can improve overall survival in patients with childhood ALL needs to be evaluated in adequately controlled clinical trials (AU)

Fluorescent In Situ Hybridization for the Detection of Intracellular Bacteria in Companion Animals.

FISH techniques have been applied for the visualization and identification of intracellular bacteria in companion animal species. Most frequently, these techniques have focused on the identification of adhesive-invasive Escherichia coli in gastrointestinal disease, although various other organisms have been identified in inflammatory or neoplastic gastrointestinal disease. Previous studies have investigated a potential role of Helicobacter spp. in inflammatory gastrointestinal and hepatic conditions. Other studies evaluating the role of infectious organisms in hepatopathies have received some attention with mixed results. FISH techniques using both eubacterial and species-specific probes have been applied in inflammatory cardiovascular, urinary, and cutaneous diseases to screen for intracellular bacteria. This review summarizes the results of these studies.