Fluorescence in situ hybridisation (FISH) detects defined DNA targets directly in fixed cells or tissue by hybridising fluorescently labelled probes to complementary sequences. A reliable FISH workflow depends on four things working together: specimen preparation, target/probe denaturation, probe-specific hybridisation, and correctly controlled post-hybridisation washing. There is no single universal temperature or incubation time for every probe and specimen; use the probe IFU or a validated laboratory method as the final authority.
Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique used to visualise specific nucleic-acid sequences inside fixed cells, nuclei, chromosome preparations or tissue sections. Depending on probe design, FISH can be used to study chromosomal rearrangements, gene fusions, copy-number changes, deletions, amplifications, aneuploidy and defined genomic loci while preserving spatial information at the single-cell level.
This SOP is written as a research-use starting framework for DNA FISH on cytological preparations and formalin-fixed, paraffin-embedded (FFPE) tissue. It is intentionally probe-agnostic. Manufacturer instructions, probe chemistry, specimen fixation and your validated scoring criteria take precedence over the general examples below.
What does a FISH assay measure?
A fluorescent probe is designed to bind a defined genomic region. After the probe and target DNA are denatured into single strands, complementary sequences hybridise. Unbound or weakly bound probe is then removed by stringency washing. The remaining fluorescent signal is examined under an epifluorescence microscope and interpreted according to the probe design and the laboratory's validated scoring criteria.
Common FISH designs include break-apart, dual-fusion, enumeration/centromeric, deletion and amplification probes.
Common specimen types
- Interphase nuclei: uncultured cells, blood or bone marrow preparations, cell pellets and tissue sections.
- Metaphase chromosome preparations: useful where chromosomal location and structural context are required.
- FFPE tissue: widely used in pathology and research, but fixation, paraffin removal and protease treatment materially affect assay quality.
- Fresh or frozen material: may require different fixation and permeability conditions from FFPE.
Equipment
- Positively charged glass slides where appropriate for tissue sections
- Calibrated pipettes suitable for low-microlitre volumes
- Coplin jars or staining dishes
- Water bath, heating block or validated hybridisation instrument
- Humidified hybridisation chamber
- Calibrated thermometer
- Forceps and solvent-resistant marker
- Fluorescence microscope with filter sets matched to the probe fluorophores and DAPI
- Low-autofluorescence immersion oil where oil objectives are used
Core reagents
- Target-specific FISH probe or probe set
- SSC-based wash solutions or the probe manufacturer's proprietary buffers
- Hybridisation buffer supplied or validated for the probe chemistry
- Formamide where required by the assay chemistry
- Graded ethanol solutions for dehydration
- DAPI or another validated nuclear counterstain
- Rubber cement or another validated coverslip sealant where required
- For FFPE: xylene or a validated substitute, graded ethanol, pretreatment buffer and a validated protease/pepsin digestion system
Before starting: control the variables that matter most
- Fixation: under- or over-fixation can change nuclear morphology, target accessibility and signal intensity.
- Pretreatment: especially important in FFPE tissue. Too little can limit probe penetration; too much can destroy morphology.
- Denaturation: insufficient denaturation reduces hybridisation; excessive heat/time can damage nuclei and increase artefact.
- Hybridisation: time and temperature are chemistry-specific. Do not shorten an overnight method simply because another manufacturer's probe supports FAST FISH.
- Wash stringency: a balance is required. Low stringency leaves background; excessive stringency can remove true signal.
- Microscope setup: filter sets, lamp/laser condition, exposure and gain can make a technically good slide appear poor.
General FISH procedure
1. Prepare the specimen
Prepare the slide according to the specimen type. Cytological preparations should have intact, well-spread nuclei with minimal debris. FFPE sections should be securely attached to positively charged slides and processed to remove paraffin completely.
2. FFPE deparaffinisation and pretreatment
- Deparaffinise FFPE slides using a validated xylene/xylene-substitute workflow and rehydrate through graded alcohol to aqueous buffer.
- Perform heat pretreatment using the probe manufacturer's validated buffer and temperature. As one example, Agilent's FISH General Purpose Reagents protocol uses pretreatment solution heated to 98°C for FFPE samples; CytoCell FFPE protocols likewise use near-boiling pretreatment, with duration adjusted to fixation and tissue quality.
- Apply the recommended protease or pepsin digestion. Begin with the product IFU condition and adjust only through controlled optimisation.
- Wash and dehydrate through graded ethanol if required by the assay.
3. Prepare the probe
- Allow the probe to reach the handling temperature specified in its IFU.
- Mix gently but thoroughly. Some products permit vortexing; others should be mixed by pipetting. Follow the product-specific instructions.
- Briefly centrifuge the tube if needed to collect contents.
- Use the recommended volume per target area. Many commercial FISH assays use approximately 10 µL per 22 × 22 mm coverslip area, but this is not universal.
4. Apply probe and coverslip
- Apply the probe to the target area without introducing bubbles.
- Place the coverslip carefully. Excess pressure can squeeze probe out from under the coverslip and produce patchy hybridisation.
- Seal the coverslip where the method requires it to prevent evaporation during denaturation and hybridisation.
5. Denature probe and target DNA
Co-denaturation is commonly performed in the low-to-high 70°C range for a few minutes, but the exact temperature and time are probe-specific. Abbott's validated IntelliFISH FFPE example uses 73°C for 5 minutes before hybridisation. Do not transfer that setting to a different probe system without validation.
6. Hybridise
Incubate in a humidified chamber at the temperature specified for the probe. Conventional commercial workflows commonly use overnight hybridisation, often around 14–24 hours. Validated accelerated systems can reduce this substantially; Abbott reports 2–3 hour hybridisation for its fast-working chemistry, Agilent's IQFISH supports 1–2 hours, and OGT/CytoCell offers validated 2-hour FAST FISH workflows for selected probes. See FAST FISH vs conventional FISH for a detailed comparison.
7. Post-hybridisation wash
- Remove the coverslip using the probe-specific wash procedure.
- Perform the validated stringency wash at the specified buffer concentration, temperature and duration.
- Avoid allowing the tissue or cell preparation to dry between critical wash steps unless the IFU explicitly instructs it.
- Protect fluorescent probes from unnecessary light exposure.
Abbott's validated fast-working FFPE example uses 2× SSC/0.3% Tween 20 and a 73°C wash for 3 minutes after an initial ambient wash. Other systems use different buffers and temperatures. Treat the IFU as authoritative.
8. Counterstain and mount
- Air-dry or rinse as directed by the assay.
- Apply DAPI/antifade counterstain.
- Coverslip and protect the slide from light.
9. Image and score
- Scan at lower magnification to identify representative, well-preserved areas.
- Score signals using the magnification, filters and criteria validated for the probe and specimen.
- Avoid overlapping nuclei, damaged nuclei, areas with excessive background and clearly truncated nuclei unless the scoring protocol explicitly addresses them.
- Use the probe map and expected normal/abnormal signal patterns. Break-apart and fusion thresholds are not universal; follow the specific IFU and your validated assay criteria.
Recommended controls
- Known positive control: confirms that the assay can detect the expected signal pattern.
- Known negative control: helps assess specificity and expected normal signal architecture.
- Probe-specific normal control: useful for establishing background split/fusion rates where applicable.
- Reagent and run controls: required according to the product, laboratory and regulatory context.
How do I know whether a FISH run worked?
A technically acceptable slide should have preserved nuclei or tissue morphology, clear DAPI counterstaining, discrete probe signals at the expected intensity, low diffuse background, and signal patterns that are interpretable under the probe-specific scoring rules. If the positive control fails, do not interpret experimental samples until the cause is resolved.
Common failure modes
| Problem | Likely variables | First checks |
|---|---|---|
| Weak/absent signal | Probe integrity, insufficient pretreatment, poor denaturation, short/incorrect hybridisation, microscope/filter mismatch | Confirm positive control; review probe storage; confirm IFU denaturation/hybridisation conditions; inspect pretreatment and imaging settings. |
| High background | Low wash stringency, excess probe, tissue autofluorescence, evaporation during hybridisation | Check wash temperature/buffer, coverslip seal, probe amount and imaging exposure. |
| Poor morphology | Over-digestion, harsh heat pretreatment, over-denaturation, poor fixation | Reduce one pretreatment variable at a time and compare against an intact control slide. |
| Patchy hybridisation | Air bubbles, probe squeezed from under coverslip, uneven tissue, evaporation | Reapply coverslip technique; ensure complete seal and even probe coverage. |
| Ambiguous pattern | Probe design misunderstood, truncation, overlapping nuclei, inadequate focus/z-plane | Review probe map/IFU; exclude unsuitable nuclei; confirm scoring criteria and controls. |
Frequently asked questions
How long does FISH hybridisation take?
It depends on the validated probe chemistry. Conventional workflows commonly use overnight hybridisation, whereas validated FAST FISH systems can reduce hybridisation to about 1–3 hours. Shorter is not automatically better: the probe, specimen and wash protocol must be validated together.
What temperature is used for FISH hybridisation?
Commercial DNA FISH workflows often hybridise around 37°C, but probe chemistry varies. Use the temperature specified in the probe IFU.
Can I use the same protocol for FFPE and cytological preparations?
No. The core hybridisation principles are the same, but FFPE usually requires deparaffinisation, heat pretreatment and carefully controlled protease digestion. Cytological preparations generally require a different pretreatment workflow.
What is the difference between break-apart and fusion FISH?
Break-apart probes flank one locus and detect rearrangement through separation of signals. Dual-fusion probes target two known partner loci and detect a specific fusion through signal co-localisation. See the FISH probe selection guide.
Explore FISH probes and technical support
ABMIUM currently lists 444 active research-use FISH Probe Reagents from Wuhan HealthCare Biotechnology within its wider FISH probe collection, covering break-apart, fusion, deletion, amplification, enumeration and locus-specific designs. If you are planning a short-hybridisation workflow, contact ABMIUM with the target, specimen type and intended workflow so we can confirm the relevant probe and manufacturer-supported conditions before ordering.
You can also browse the Wuhan HealthCare collection or use Find your MATCH for help narrowing the probe design.
References and technical sources
- NHS Genomics Education Programme — Fluorescent in situ hybridisation (FISH).
- Abbott Molecular — Universal Pretreatment and Hybridization Conditions for FFPE Specimens.
- Agilent — FISH General Purpose Reagents Protocol.
- OGT/CytoCell — Example haematology FISH protocol.
- Speicher MR, Carter NP. Fluorescence in situ hybridization: cell-based genetic diagnostic and research applications.