Several rounds of thorough washing for the appropriate amounts of time are also critical for low fluorescent backgrounds this is not a place to cut corners or time. Improper storage of the slides after opening the foil pouch may also cause an uneven background. Be sure to reseal the unused slides in their storage containers immediately after removing the slides to be used for printing. In protocols where no blocking step is used, nonspecific binding to the slide surface can result in high and variable background levels. Entrapment of air bubbles underneath the cover slip can prevent the cDNA probe solution from contacting parts of the slide surface.
Bubbles can result in varying levels of background across the slide surface.
Dirty coverslips can cause bubble entrapment. I see negatively stained spots in the background. It looks like the coating has come off the slide. This occurs when the probe binds nonspecifically to the background during the hybridization step. As the unexpressed DNA is already completely bound to its area, the probe solution does not bind nonspecifically to this area.