RNA whole mount in situ hybridization of 3 dpf embryos with dat probe didn′t show significant loss of DA neurons in morphant.

E to H, L and M, enlargement of the area of DA neurons in A to D, J, and K, respectively. The numbers on bottom right corner showed number of the embryos with a certain phenotype/number of total embryos in that group. The patterns of DA neurons in most embryos were normal, while some were disorganized. I, the quantitative result of dat positive neurons in the diencephalon. There is no significant DA neuron loss in morphant group. n = 20 in each group, P>>0.05 in all comparisons.

RNA whole mount in situ hybridization of 3 dpf embryos with th probe didn′t show significant loss of DA neurons in LRRK2 morphants.

A to D, the diencephalon region of zebrafish embryos, dorsal view, anterior to the top. The numbers on bottom right corner showed number of the embryos with a certain phenotype/number of total embryos in that group. There was not obvious alteration of DA neurons pattern in the diencephalon of the morphants (B, C, and D). E, quantative result of th positive neurons in the diencephalon. There was not significant DA neurons loss in all three morphant groups. n = 20 in each group, P>>0.05 in all comparisons.

Flatten embryo and count DA neurons. Take embryos hybridized with dat probe as an example. After in situ hybridization, transfer embryos into glycerol and equilibrate for 10 min. Then put it on a slide and flatten it softly with a cover slip to disperse the neurons (A, dosal view; B, lateral view). C and D show the same embryo before and after being flattened.

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