It is frequently suggested that parts of the protocol for FISH on cytogenetic samples have to be done in high relative humidity conditions. According to your opinion and knowledge, what is the real/deep motivation for that?
It is not clear to me at which step you are asking the humidity.
If it concerned incubation step during FISH, it would be to avoid loss of you probe or antibody mixes at the detection steps. However, for FISH there are good automated systems and then you can see, well sealed chamber for hybridization and other wet incubation provided by the machine does the job.
Is this what you were looking for?
Best regards,
Karoly
Humidity in cytogenetic FISH is crucial in two steps of the procedures- when you leave the slide overnight at 37 degree Celsius in dark chamber, and the other day when you remove the coverslip till placing the slide inty washing solution, the slide shouldn't dry
Actually, after you add the hybridization mix you should not leave the preparations get dried, because this will interfer in the hybridization reaction of probe to DNA.
Humidity is required while making slides and probe hybridization. Humidity for slidemaking is required mostly for FISH slides done from peripheral blood samples. We almost never use a steamer for preparing bone marrow slides.
Apart from that we prepare a humidity chamber for incubating FISH slides for probe hybridization. We just line wet tissue on the bottom of a slide box, arrange the slides over it and keep it at 37 degrees overnight. We do have a thermobrite in which we keep wet strips of Whatmann paper, but it can hold only 12 slides. On the days we have more cases , we use the humidity box. Hope this helps.
Humidity is necessary for making slides and probe hybridization from peripheral blood
Slide making has been covered above, so I'm just going to address the need for humidity in the hybridization chamber. Humidity is important to maintain the correct formamide concentration in the probe solution. Sealing the cover slip helps, but if rubber cement is used, it can absorb water from the probe solution. Thus, keeping the hybridization chamber at a higher RH, will keep the rubber cement from absorbing water. However, if the chamber has an RH that is too high, the rubber cement can absorb too much water, and the probe mixture may then absorb the excess water in the rubber cement. Formamide concentrations that are too high or too low can yield weaker FISH signals and/or higher background/non-specific binding. The ideal RH is ~50%. There are cover slip sealing products you can use that don't require a humidified chamber.
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