Hematology FISH Protocol

Hematology fluorescence in situ hybridization (FISH) is a powerful cytogenetic technique that is widely used in the diagnosis, management, and monitoring of hematological malignancies, such as leukemia, lymphoma, and myeloma. This method allows for the detection and visualization of specific chromosomal abnormalities that are often associated with these blood-related cancers.

Sample and slide preparation

• Spot the cell sample onto a glass microscope slide. Allow to dry.
• Immerse the slide in 2xSaline Sodium Citrate (SSC) for 2 minutes at room temperature (RT) without agitation.
• Dehydrate in an ethanol series (70%, 85%, and 100%) for 2 minutes at RT. Allow to dry.

Pre-denaturation

• Remove the probe from the freezer and allow it to warm to RT. Briefly centrifuge tubes before use.
• Ensure that the probe solution is sufficiently mixed with a pipette or a vortex mixer.
• Remove 10 μl of probe per test, and transfer it to a microcentrifuge tube. Quickly return the remaining probe to -20ºC.
• Place the probe and the sample slide to prewarm on a 37°C (+/- 1°C) hotplate for 5 minutes.
• Spot 10μl of probe mixture onto the cell sample and carefully apply a 24x24mm coverslip. Seal with rubber solution glue and allow the glue to dry completely.

Denaturation

• Denature the sample and probe simultaneously by heating the slide on a hotplate at 75°C (+/- 1°C) for 2 minutes.

Hybridization

• Place the slide in a humid, lightproof container overnight at 37°C (+/- 1°C).

Post-hybridisation washes

• Remove the DAPI from the freezer and allow it to warm to RT.
• Remove the coverslip and all traces of glue carefully.
• Immerse the slide in 0.4xSaline Sodium Citrate (SSC) (pH 7.0) at 72°C (+/- 1°C) for 2 minutes without agitation.
• Drain the slide and immerse it in 2xSSC + 0.05% Tween-20 at RT (pH 7.0) for 30 seconds without agitation.
• Drain the slide and apply 10 μl of DAPI antifade onto each sample.
• Cover with a 24x24mm coverslip, and remove any bubbles.
• Edge the slide with clear nail varnish to seal.
• Allow the color to develop in the dark for 10 minutes.

Analyze

• View with a fluorescence microscope.
• For optimal visualization of the probes, a 100-watt mercury lamp (or equivalent) is recommended with plan apochromatic objectives 63x or 100x.
• Filters designed specifically for the detection of DAPI and FITC, individually or in combination (e.g., dual or triple filters) are optimal for best results.

Creative Bioarray Relevant Recommendations

• Creative Bioarray offers a range of different FISH services including metaphase and interphase FISH (chromosomal assignment and clone ordering), Fibre-FISH (Chromosome Painting), RNA-FISH (cell-based gene expression assay), M-FISH (multicolor karyotyping), 3D-FISH (on three-dimensionally preserved nuclei), Flow-FISH (quantify the length of telomeres), FISH on paraffin sections (analysis of archive material), ImmunoFISH (combined FISH and IHC).
• We also provide the most comprehensive list of FISH probes to rapidly identify a wide range of chromosomal aberrations across the genome.

• Proper cell culture and fixation techniques are crucial to obtaining well-spread, morphologically intact metaphase chromosomes or interphase nuclei.
• Choosing the appropriate FISH probes that target specific chromosomal regions or genetic alterations associated with hematological malignancies is essential.
• The probes should be designed and labeled with fluorescent dyes that provide clear, distinct signals for accurate interpretation.

Fluorescent In Situ Hybridization (FISH) Service

For research use only. Not for any other purpose.