When scanning my microarrays a shadow appeared in the Cy3 Channel (see picture below). I used a mouse microarray from Microarray Inc. and pre-hybridized it. I also washed and dried the array carefully.
The shadow only happens in the Cy3 channel, but not in the Cy5 channel.
Any idea of why this is happening and how solve it?
Hi Sebastian,
How are you hybridizing the array? There is an excess of Cy3 all over the slide, although it is most prevalent near the bar code. It is difficult to tell for certain, but it looks like parts of the array may have dried or formed bubbles during the hybridization. As such, it is almost impossible to wash all of this off. This has also happened to me when using a Tecan automated hybridization station. The problem was similar - a broken gasket caused parts of the slide to dry and not be washed. Is this an isolated problem or are all your slides like this?
I put the hybritadion mix over the array and cover it up with a glass slide and close the chamber. Then dive the chamber in a thermoregulated bath for 21 hour at 34°C.
The chamber is removed from the water and the array is washed( The Slide fall off during the first wash)