Sectioning of zebrafish larvae is better than the whole-mount method
posted by: acarr
John G. joined the lab a few weeks ago to work out the whole-mount immunolabeling of our wild-type and mutant fish. Thus far, we have had problems with too much fluorescent background, mixed with our specific signals. We see the dopaminergic cells, but we also see everything else in red color (although dimmer than the color in the dopaminergic cells).
So, I decided to make some cross-sections of fixed embryos/larvae, to see if the fluorescent labeling method would be better. The problem with sectioning is that the embryos/larvae are so small. You have to position them just right to get good orientation in sectioning. But, if you section enough, you will get a few really good sections. John G. did the immunolabeling of the cross-sections.
Here are images of the tyrosine-hydroxylase labeling of the dopaminergic cells in 3 day old larvae (green fluorescent color).
These are normal (non-mutant) fish. The signal is clearer, and there is minimal background. Note the labeling in the eyes and brain (area between eyes), and some cell axons. The cells bodies are bright green. Depending on the cross-section, you will get good orientation of the retina in the eye (with lens). Not every cross-section is good.
I sectined ~15-20 larvae to get these pics. (I put about 10 larvae in an ice block, and section all of them at the same time…takes me about 15 min per block).