What are the factors that influence Fluorescence in situ hybridization (FISH) performance?

Factors such as temperature, buffer composition, salt concentration, and hybridization time can influence the efficiency and specificity of FISH. 

1. Optimal hybridization conditions should be determined individually for each FISH assay. Temperature plays a crucial role in FISH hybridization as it directly affects the kinetics of probe-target binding. 
2. Generally, a higher temperature increases the rate of hybridization but may also lead to non-specific binding. In contrast, a lower temperature reduces the rate of hybridization but may enhance specificity. 
3. In standard FISH protocols, the hybridization process typically occurs at a temperature of 46°C for a duration of 2 to 3 hours. 
4. The salt concentration is maintained at a constant level, typically consisting of 750 mM NaCl and 87.5 mM sodium citrate. Additionally, the design of the FISH probe (such as length and specificity) impacts its performance. The goal of FISH probe design is to achieve a balance between sensitivity to the target sequences and specificity to avoid cross-hybridizations with unrelated sequences.