Protocol for Fluorescence In Situ Hybridization (FISH)
Materials Required
Principle
Fluorescence in situ hybridization detects specific DNA or RNA sequences inside fixed cells or tissue sections by hybridizing fluorescently labeled nucleic-acid probes to complementary target sequences, allowing the target’s copy number, chromosomal position, spatial distribution, or transcript abundance to be visualized microscopically[1][2][3].
DNA-FISH detects genomic loci, chromosomal gains/losses, amplifications, deletions, and rearrangements, while RNA-FISH detects RNA molecules or transcript localization; in cancer cells, mouse tumors, neurons, organoids, macrophages, or drug-screening samples, the readout is fluorescent puncta, fusion/split signals, or localized RNA signal interpreted relative to validated controls[2][4][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
Reagents and chemicals
• Use fixed cultured cells, cytospins, touch preparations, frozen sections, or formalin-fixed paraffin-embedded tissue sections as sample formats; fixation preserves morphology while allowing probe access to DNA or RNA targets[4][6][7].• Use formamide-containing hybridization buffer, saline-sodium citrate wash buffer, ethanol dehydration solutions, and nuclear counterstain such as DAPI for DNA-FISH workflows involving denaturation and post-hybridization washing[2][4][6].
Antibodies, probes, dyes, or kits
• Use locus-specific, centromeric, break-apart, fusion, chromosome-painting, or oligonucleotide probe sets according to whether the target is copy number, translocation, chromosome structure, or RNA abundance[2][4][5].• Use immunofluorescence-compatible antibodies only when combined RNA-FISH or DNA-FISH with protein localization is required, because combined FISH/immunofluorescence has been described for tumor and cellular marker analysis[7][8].
Cells, tissues, isolated organs, organoids, or animals
• Use cancer cells, primary neurons, mouse tumor sections, intestinal organoids, inflammatory macrophages, or drug-treated cultures as fixed biological specimens; dissociated organoid cells or organoid sections are preferred when single-cell signal quantification is required[3][5][8].Buffers and solutions
• Prepare fixation, permeabilization or pretreatment, denaturation, hybridization, wash, blocking, and mounting solutions matched to the probe type and sample format; FFPE tissues often require pretreatment to improve probe penetration[4][6][7].Equipment and instruments
• Use a humidified hybridization chamber, temperature-controlled slide denaturation/hybridization system or incubator, fluorescence microscope or confocal microscope, appropriate filter sets, cooled camera, image-analysis software, and flow cytometer only when performing FISH-Flow[2][5][9].Controls
• Include positive-control cells or tissues known to contain the target, negative-control specimens lacking the target, no-probe controls, scrambled or irrelevant probes for RNA-FISH, nuclear counterstain, and sample-specific cutoff thresholds for clinical-style signal interpretation[4][6][10].Experimental Procedure
Preparation Steps
• Prepare samples by fixing cultured cells, tissue sections, cytospins, or organoid-derived cells using a method compatible with the selected DNA-FISH or RNA-FISH probe system; preserve comparable fixation and processing across all experimental groups, including vehicle and drug-treated samples[3][6][7].• Select probes according to the biological question: use DNA-FISH probes for HER2 amplification, BCR-ABL fusion, ALK rearrangement, chromosomal copy number, or tumor cytogenetics; use RNA-FISH or smFISH oligonucleotide probe sets for transcript localization or single-cell RNA quantification[2][4][5].
• For combined FISH with immunofluorescence, choose fixation and permeabilization conditions that preserve both nucleic-acid signal and antigen detection, because combined FISH/immunostaining has been used to assign nucleic-acid signals to specific tumor or microenvironment cells[7][8].
Operation Steps
• Mount fixed cells or tissue sections on slides, perform sample pretreatment or permeabilization appropriate for the specimen, and dehydrate when required by the selected FISH method[4][6][7].• For DNA-FISH, denature sample DNA and probe DNA, apply probe to the specimen, coverslip, and hybridize under probe-specific conditions; exact temperature and time must follow the validated probe protocol because these parameters vary across probe chemistry and tissue format[2][4][6].
• For RNA-FISH, avoid DNA denaturation unless required by the method, hybridize fluorescent oligonucleotide or RNA probes to fixed permeabilized samples, and maintain RNA-preserving conditions throughout processing[3][5][9].
• Wash slides under stringency conditions matched to probe design, target sequence, and hybridization chemistry to reduce nonspecific binding while retaining target-specific signal[2][4][5].
• Counterstain nuclei with DAPI or an equivalent nuclear dye, mount in antifade medium, and image using fluorescence channels appropriate for the fluorophores used[2][4][5].
• For FISH combined with immunofluorescence, perform antibody staining in the validated order for the chosen protocol and acquire both nucleic-acid and protein-marker channels under matched imaging settings[7][8].
• For FISH-Flow, fix and permeabilize nonadherent cells, hybridize fluorescent oligonucleotide probes, optionally stain proteins with fluorescent antibodies, and quantify mRNA and protein simultaneously by flow cytometry[9].
Data Acquisition and Analysis
• Acquire images using identical microscope settings across comparable samples, avoiding saturated pixels, and score only nuclei or cells with interpretable morphology and adequate probe signal[2][4][10].• For DNA-FISH, quantify signal number, fusion/split pattern, copy-number ratio, or percentage of abnormal cells; define assay-specific positivity thresholds using control specimens because nonspecific or background signals can occur in a small fraction of cells[4][6][10].
• For RNA-FISH or smFISH, quantify fluorescent puncta per cell, total cellular intensity, or subcellular localization; normalize to cell number, tissue region, treatment group, or protein-marker-defined cell population when combined with immunofluorescence or flow cytometry[3][5][9].
• Use independent biological samples for comparisons among cancer cells, neurons, tumor tissue, organoids, macrophages, or drug treatments, and include technical replicate fields, sections, or wells to estimate imaging and scoring variability[5][9][10].
Troubleshooting
Problem: Weak or absent FISH signal.
Possible Cause: poor probe hybridization, degraded target, inadequate pretreatment, or over/under-processing of FFPE tissue.Literature-supported Solution: use validated positive-control specimens, optimize tissue pretreatment for FFPE samples, and confirm that probe and sample format are compatible[4][6][7].
Problem: High background fluorescence.
Possible Cause: nonspecific probe binding or insufficient stringency washing.Literature-supported Solution: adjust hybridization and wash stringency within the validated range for the probe system and include no-probe or irrelevant-probe controls[2][4][5].
Problem: Ambiguous copy-number interpretation.
Possible Cause: truncation of nuclei in tissue sections, polysomy, or overlapping cells.Literature-supported Solution: score adequate numbers of intact interpretable nuclei, use paired control probes when appropriate, and define laboratory- or study-specific thresholds from controls[4][6][10].
Problem: RNA-FISH signal is lost after immunofluorescence.
Possible Cause: incompatible fixation, permeabilization, or antibody-staining sequence.Literature-supported Solution: use a protocol specifically validated for combined mRNA FISH and immunofluorescence[7][8].
Problem: Flow-cytometry FISH signal overlaps with autofluorescence or weak expression.
Possible Cause: low transcript abundance or insufficient probe signal.Literature-supported Solution: use multi-probe RNA-FISH probe sets, matched negative controls, and gating strategies validated in FISH-Flow workflows[9].
References:
- [1]. Pinkel D, et al. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A. 1986;83(9):2934-2938. [Content Brief]
- [2]. Speicher MR, et al. The new cytogenetics: blurring the boundaries with molecular biology. Nat Rev Genet. 2005;6(10):782-792. [Content Brief]
- [3]. Femino AM, et al. Visualization of single RNA transcripts in situ. Science. 1998;280(5363):585-590. [Content Brief]
- [4]. Nolte M, et al. Fluorescence in situ hybridization is a reliable diagnostic tool for detection of the 9;22 translocation. Leuk Lymphoma. 1996;22(3-4):287-294. [Content Brief]
- [5]. Raj A, et al. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008;5(10):877-879. [Content Brief]
- [6]. Persons DL, et al. Fluorescence in situ hybridization for detection of HER-2/neu amplification in breast cancer: a multicenter portability study. Ann Clin Lab Sci. 2000;30(1):41-48. [Content Brief]
- [7]. Kiprianova I, et al. Fluorescence in situ hybridization detection of viral nucleic acids in oncolytic H-1 parvovirus-treated human brain tumors. Methods Mol Biol. 2020;2058:295-306.
- [8]. Gasnier M, et al. Fluorescent mRNA labeling through cytoplasmic FISH. Nat Protoc. 2013;8(12):2538-2547.
- [9]. Arrigucci R, et al. FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc. 2017;12(6):1245-1260.
- [10]. Belaud-Rotureau MA, et al. Interphase fluorescence in situ hybridization is more sensitive than BIOMED-2 polymerase chain reaction protocol in detecting IGH-BCL2 rearrangement in both fixed and frozen lymph node with follicular lymphoma. Hum Pathol. 2007;38(2):365-372. [Content Brief]
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