How can I fix yolk staining in zebrafish embryo immunofluorescence?

[MarkET]

I've been trying to map the expression of a specific transcription factor in my zebrafish embryo samples, but my immunofluorescence keeps coming back with this weird, non-specific staining in the yolk. I'm worried my antibody concentration is too high, but when I lower it, the signal in the actual tissue becomes too faint to quantify properly.

[JeffreyTW]

Yeah I ran into this too. My immunostaining would light up the yolk like a ticker tape and the tissue signal vanished when I lowered the antibody too much. I ended up stuck between a wall of non specific yolk signal and a weak target signal in the embryo. It felt like chasing ghosts.

[Chloe.G]

Could yolk autofluorescence be masking the real signal too? In my hands, the yolk glowed in the same channels regardless of what I did to the stain. I started using a different fluorophore window and looked at edges of the embryo rather than center; still it comes back.

[Kevin14]

I did try a longer blocking and a different blocking agent and a different secondary, but the background stayed. I measured signal in a control zone and it was not changing even when the tissue signal looked good. It was frustrating to quantify.

[Thomas91]

Sometimes I wonder if the whole setup is overthinking the biology here and maybe the transcription factor just isn't there at the time point. Not sane to think that, but I keep staring at the prep and wondering if I'm barking up the wrong tree.