What can cause false positives in zebrafish embryo in situ hybridization?

[IsabellaA]

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 positive signal that doesn't reflect the actual gene activity pattern.

[ZoeyMW]

I had that diffuse look once. I compared the probe sequence with similar genes and found a close family member. I redesigned to target a unique region and used a longer probe. The signal shifted and the background dropped.

[MichaelOM]

Maybe the problem is not the gene but the protocol. Fixation and washing can leave tissue sticky. I would run a sense control to see if the staining disappears. If it stays the same I would suspect non specific binding.

[NatalieUW]

I'm not sure this is the real problem. The issue cycles in my work and I try tool changes but still not clean. I lowered stringency a bit and saw more background. I raised stringency and got worse image.

[Jeffrey60]

I spent a day chasing a tangential thought about the stage adaption of embryos. Then I came back to the probe issue. The stage affects background intensity and the same probe behaved differently in slightly older embryos.