Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique that detects and localizes specific nucleic acid sequences within fixed cells or tissue sections using fluorescently labeled probes. The hybridization buffer plays a crucial role in ensuring efficient, specific, and stable binding of these labeled probes to their complementary target sequences during the hybridization step. The optimal composition of the hybridization buffer affects signal intensity, background noise, hybridization speed, and preservation of cellular morphology.

Mechanism of Action

Formamide disrupts hydrogen bonds between DNA strands, effectively lowering the melting temperature and allowing hybridization to occur efficiently at ~37–42°C rather than higher temperatures that could damage sample morphology.

Dextran sulfate increases the viscosity of the hybridization mix, so probes are concentrated near the target sequences, speeding the hybridization rate.

SSC maintains salt conditions critical for nucleic acid duplex stability — higher salt stabilizes the duplex, whereas lower ionic strength increases stringency during post-hybridization washes.

SDS reduces hydrophobic interactions between the probe and non-target molecules, increasing hybridization specificity.

Hybridization buffers for Fluorescence In Situ Hybridization are carefully formulated solutions composed primarily of formamide, dextran sulfate, saline sodium citrate buffer, and SDS to enable efficient, specific hybridization of fluorescent probes to target DNA or RNA sequences. Innovations in hybridization buffer chemistry—such as the inclusion of alkyl diesters—aim to reduce nonspecific binding and improve assay robustness. Optimization of buffer composition, concentration, and hybridization parameters is essential for achieving high-quality, reproducible FISH results in research and clinical diagnostics.