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Analysis

How should slides that have been FISH’d be stored?

Once the DAPI counterstain and coverslip have been applied to the slide, it is recommended that users seal the coverslip with varnish. This prevents excessive drying out and maintains the probe signal strength. For short term storage 0-2 weeks the slides should be covered and stored at room temperature. For long term storage, the slides should be stored 2-8°C and covered.

What are the correct filter characteristics that would allow for simultaneous visualization of red, green and aqua?

Currently, there are two different triple filters in common use in laboratories, one which is specific for TRITC/FITC/DAPI and one which allows visualization of Texas Red/FITC/DAPI. The first one is easily distinguished by the green light that comes from the objective on the microscope, the other gives a white/pale yellow color. The only filter that allows simultaneous visualization of Cytocell's green, red and blue dyes is a Texas Red/FITC/DAPI filter.

Can spectrum orange filters be used to visualize Cytocell Texas Red probes?

Both types of filter can be used, but using the Spectrum Red will give brighter signals for Texas Red probes and using the Spectrum Orange will give brighter signals for our orange fluorophore.

‘We are using a very old filter and we suspect that it causing problems during analysis. How do you identify a poor filter? What effect does this have?’

Depending on the manufacturer’s recommendations, fluorescent filters typically need to be replaced every 2-4 years. Over time and use, the filter will begin to degrade. The damaged emission/excitation filters will show a mottled appearance, which begins on the edges and will eventually completely obscure the filter. The effects may not be noticeable on a day to day basis, but will manifest as higher than normal background and weaker signals.

Please contact Cytocell Technical support for further advice and guidance if you suspect filter problems.

How long do the high-pressure mercury microscope bulbs last for?

Depending on the type of bulb used, the shelf life can vary from 200-3000 hours, please verify with your supplier. The older the bulb, the dimmer the fluorescence signals become, and the greater the chance of the bulb exploding. This is dangerous as mercury vapor is highly toxic.