In Situ Hybridization (ISH) Service: Accurate, Reliable, and Tailored for Your Research
Unlock the full potential of spatial biology with Creative Proteomics' premier In Situ Hybridization (ISH) services. We specialize in the precise localization of specific DNA and RNA sequences within preserved tissue architectures, bridging the critical gap between molecular genomics and cellular morphology. By combining rigorous assay optimization with advanced detection platforms, Creative Proteomics empowers researchers to visualize gene expression and chromosomal aberrations with the sensitivity, specificity, and spatial resolution required for impactful scientific discovery and clinical development.
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- Our service
- Workflow
- Why choose
- Deliverables
- Applications
- FAQs
- Sample preparation
Our Service
We offer end-to-end ISH solutions, from custom probe design to high-throughput slide scanning and quantitative image analysis. Our laboratory is equipped to handle difficult tissue matrices, including FFPE (Formalin-Fixed Paraffin-Embedded), frozen tissue, and cell microarrays.
- Fluorescence In Situ Hybridization (FISH)
Leverage our high-sensitivity FISH protocols for multi-target detection. We utilize spectrally distinct fluorophores to visualize multiple genetic targets simultaneously, providing a robust solution for:
- Gene Amplification & Deletion: Precise copy number variation (CNV) analysis (e.g., HER2, MET).
- Chromosomal Translocations: Detection of gene fusions and rearrangements (e.g., ALK, ROS1).
- Subcellular Localization: High-resolution mapping of nucleic acids within specific cellular compartments.
- Chromogenic In Situ Hybridization (CISH)
For studies requiring simultaneous evaluation of tissue morphology and gene status, our CISH services utilize enzyme-labeled probes (HRP/AP) to generate permanent, bright-field compatible signals.
- Pathology-Friendly: Allows for analysis using standard light microscopes.
- Morphological Context: Excellent retention of tissue architecture for identifying cell-type-specific expression.
- Archival Stability: Permanent staining allows for long-term storage and re-analysis of slides.
- Silver-enhanced In Situ Hybridization (SISH)
Our SISH services utilize enzyme-metallography to deposit silver particles at the target site, generating discrete, permanent signals that allow for precise gene copy quantification without the need for fluorescence.
- High-Contrast Visualization: Generates sharp, black signals against the tissue background.
- FISH-Like Sensitivity: Delivers detection limits comparable to fluorescence methods while maintaining the workflow simplicity of standard immunohistochemistry.
- Permanent Archiving: Silver signals are permanent and resistant to fading, allowing for indefinite slide storage and re-analysis.
The general workflow
Why Choose Creative Proteomics for ISH Services?
- Specialized Expertise in Challenging Samples: Our lab is uniquely equipped to handle difficult matrices, including archived FFPE blocks, frozen tissues, and cell microarrays.
- Custom Probe Design & Bioinformatics: We offer custom probe design for novel targets and non-model organisms, using bioinformatics to ensure high specificity and minimal off-target binding.
- High-Sensitivity Assay Optimization: We utilize advanced detection platforms to visualize low-abundance targets that are often undetectable by standard methods.
- Expert Technical Support: Our specialists provide pre-project consultation on sample preparation (e.g., embedding strategies and fixation) to maximize data quality.
Deliverables
Our deliverables are designed to meet the rigorous standards of high-impact scientific journals, providing you with everything needed for immediate publication.
- Publication-Quality Images: High-definition, high-contrast digital slides optimized for direct use in article figures and presentations.
- Quantitative Image Analysis: Comprehensive assessments of signal intensity and spatial localization, providing the quantitative data required for scientific discovery.
- Detailed Technical Reports: A full summary of customized protocols, probe parameters, and rigorous quality control results to ensure data reproducibility.
- Raw Data Access: All raw data files are provided for independent secondary analysis or archival purposes.
Key Applications
- Oncology & Immuno-oncology: Assess tumor heterogeneity, immune cell infiltration, and checkpoint marker expression within the tumor microenvironment (TME).
- Neuroscience: Map complex gene expression patterns in heterogeneous neural tissues.
- Infectious Disease: Localize viral or bacterial reservoirs in latent infections with high specificity.
- Developmental Biology: Track lineage-specific gene expression during embryogenesis, particularly valuable for investigating embryonic development in model organisms like zebrafish.
FAQs
Do you offer custom probe design for non-model organisms or novel targets?
Yes. We specialize in custom probe design for targets where commercial probes are unavailable. Our bioinformatics team analyzes your target sequence against the relevant genome assembly to ensure minimal off-target binding. We can design probes for virtually any species, including viral, bacterial, and rare animal models.
How does ISH differ from Immunohistochemistry (IHC)?
While both techniques visualize targets in tissue, IHC detects proteins, whereas ISH detects nucleic acids (DNA or RNA). ISH is preferred when:
- No reliable antibody exists for the protein target.
- You need to distinguish between specific gene family members with high protein homology.
- You are studying non-coding RNAs (miRNA, lncRNA).
- You need to validate gene amplification or viral infection definitively.
Can you perform ISH and IHC on the same slide (Dual-Staining)?
Yes, we offer Dual ISH-IHC services. This allows for the simultaneous visualization of RNA/DNA and protein in the same tissue section. This is particularly powerful for characterizing the cell type producing a specific cytokine or viral transcript.
Why is my signal weak or absent?
Common causes include:
- Over-fixation: Fixation in formalin for >48 hours cross-links the tissue too heavily, preventing the probe from reaching the target. (We can attempt to rescue this with stronger protease digestion).
- RNA Degradation: The tissue sat too long before fixation (ischemia time).
- Low Expression: The target may be expressed at levels below the threshold of detection for standard CISH. In these cases, we recommend our High-Sensitivity RNA amplification assays.
My samples have been stored in paraffin for several years. Are they still usable?
- For DNA ISH: Yes, DNA is highly stable. Archives >10 years old often yield excellent results.
- For RNA ISH: It depends on the fixation quality. RNA degrades slowly over time even in blocks. We strongly recommend running a RNA Quality Control check (using a housekeeping gene probe) on a subset of your blocks before proceeding with the full study to ensure the RNA is sufficiently intact.
Learn about other Q&A.
Sample Requirements
The success of in situ hybridization techniques highly depends on the quality of sample preparation. Improper sample handling can lead to nucleic acid degradation, morphological damage, failure of probe penetration, or high non-specific background signals, ultimately resulting in experimental failure.
Please share these guidelines with your lab team to minimize degradation and ensure optimal signal-to-noise ratios.
- Formalin-Fixed Paraffin-Embedded (FFPE) Samples
Standard for most CISH and FISH applications.- Fixative: Use fresh 10% Neutral Buffered Formalin (NBF). Avoid unbuffered formalin, as acidic conditions can degrade nucleic acids.
- Fixation Time:
- Optimal: 12–24 hours at room temperature.
- Critical Note: Do not over-fix (>48 hours), as this creates excessive cross-linking that masks targets. Do not under-fix (<10 hours), as this allows RNA degradation and poor morphology.
- Tissue Size: Trim samples to a thickness of 3–4 mm before fixation to ensure the formalin penetrates to the center of the tissue quickly.
- Decalcification: If analyzing bone, use EDTA-based decalcifiers. Avoid strong acid decalcifiers (e.g., HCl), which hydrolyze DNA/RNA and ruin ISH signals.
- Slide Submission:
- If sending unstained slides, section tissue at 4–5 µm thickness.
- Mount on positively charged slides to prevent tissue detachment during harsh heating steps.
- Recommendation: Send the paraffin block if possible, so we can freshly section immediately prior to the assay.
- Fresh Frozen Samples
Preferred for highly sensitive RNA ISH or targets sensitive to formalin.- Freezing Method: Flash-freeze tissue immediately after harvest using liquid nitrogen or isopentane/dry ice slurry. Slow freezing causes ice crystal formation that destroys tissue morphology.
- Embedding: Embed in OCT compound (Optimal Cutting Temperature). Ensure the tissue is completely surrounded by OCT to prevent freeze-drying.
- Storage: Store blocks at -80°C immediately. Do not allow temperature fluctuations.
- Sectioning: If you must section in-house, cut at 10–12 µm, mount on Superfrost Plus slides, and store slides at -80°C.
