Humidity during FISH probe addition

[Emma Foster]

Hi all,

I'm currently trying to figure out what effect room humidity has during the addition of the FISH probe.

I know that during cell dropping, humidity is important to allow for proper cell spreading (we do this in a Thermotron), and that during overnight hybridisation it's important to enclose the slides in a humidity box to prevent water in the probe buffer from evaporating*

However, what about when you have your slides ready, and you are adding your FISH probe to them, adding your coverslip, sealing it, and placing it on the hot plate? Has anyone noticed if lab humidity affects this process?

And what about the next day, when you take coverslips off, wash in wash buffers, add DAPI and coverslip? Any effect of humidity then?

At a biophysical level, I imagine high humidity may dilute the probe a little, but this must be by a miniscule percent, surely?

Any tips, tricks or anecdotes much appreciated! UK is going through a heatwave at the moment, so lab humidity is extreme - keep cool everyone!

All the best,

Emma

*NB: The overnight humidity box is still a bit of a scientific mystery to me. Why humid? Does the rubber cement/glue seal allow water permeabilisation? So a dry 37oC incubation would result in all the water coming out of the probe buffer? Has this ever been quantified?
Also, why does it take 16-18 h for FISH probes to find complementary sequences and bind sample DNA? This seems like a very long time. I know 2-4 hour FISHes can also be done with certain products. Just wondering about the kinetics of it all...

[Jasen Anderson]

Hi Emma, 

In our Australian lab, we use to use rubber cement to seal coverslips, but abandoned it years ago to simply place it over the probes on the slide, whether using plain glass or Teflon coated well slides. In the air conditioned environment we work in, humidity is generally low so no real effect on the process, and I imagine for the time spent adding the probe and doing the denaturation step, the effect of any humidity would be minimal, and we hyb in a humidified incubator with other closed culturing vessels, in small Kartell slide boxes (see image) with the slides laid flat across the ribs in the box, not as pictured. We have no issues with this process other than tissue or suspension dependant. 

I suspect there is very little impact with some humidity either before or after the entire process. 

Cheers

Jasen Anderson

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[Anastasia Fito]

Hello Emma,  

We have noticed that weather-related humidity have some influence on FISH reactions. As we work with histology FFPE samples, the procedure involves the usage of various alcohol reagents (xylen, ethanol), which can be affected by overall humidity. In our point of view, high humidity can reduce their effectiveness slightly and influence the quality of procedure.  

Also the procedure involves both steps that require humidity and steps where the slides must be completely dry. It is important to ensure proper drying at those stages, but high overall humidity in the lab can affect this.

To manage this, we are regulating humidity in our lab with a dry air conditioning system. In addition, we try to replace reagents more frequently than usual to maintain the quality.

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