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I've been trying to map the expression of a specific transcription factor in my zebrafish embryos, but my in situ hybridization keeps giving me this diffuse, non-specific staining. I’m worried my probe might be binding to similar mRNA sequences from other gene family members, creating a false signal.

I ran into this last year with a transcription factor. The staining looked diffuse and I worried about paralog cross talk. I trimmed the probe to a shorter region and bumped up the wash stringency. It helped a bit, but the signal never cleanly separated from background.

I dug into the sequence across the gene family and tried designing a tiled probe set so I could see which tile lit up. The idea was to map cross-hybridization, but the results were still muddy and the cross signals stuck around.

Sometimes the issue was technical—fixation, permeabilization, or enzyme diffusion. I had one batch where over-permeabilized embryos bled background staining, and the controls never turned fully blank. That was a red herring I almost ignored.

Could there be a better way to distinguish paralogs without hunting for a unique probe?

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