Journal: Scientific reports
Article Title: Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.
doi: 10.1038/s41598-023-44093-8
Figure Lengend Snippet: Figure 5. Loss of Igsf3 impairs neural crest cell migration. (A–J) E9.0 WT and KO vagal (collected at somite level 1–7) and trunk (collected at somite level 9–12) neural tube explants were cultured on fibronectin-coated cover slips for 48 h after which they were imaged. (A,B) Vagal neural crest explants isolated from WT (A) and KO (B) embryos. (C,D) Panels show the zoomed-in view of the boxed areas in (A) and (B). (E,F) Trunk neural crest explants isolated from WT (E) and KO (F) embryos. (G,H) Panels show the zoomed-in view to the boxed areas in (E) and (F). (I,J) Quantification of the neural crest cell migration. The area containing the migrating cells is marked with a green line, and the migration distance between the outer edges of the halo and the explant (black) was measured. The vagal neural crest cells of the KO explants migrated significantly less than the WT neural crest cells (I) while no difference was detected in the trunk neural crest cell migration between the WTs and KOs (J) (N = 4 for each genotype). (K,L) Representative images of the dorsal root ganglia in WT (K) and KO (L) pups at P12.5 (N = 3 for each genotype). Neurons are visualized by Tuj1-staining (green) and nuclei with DAPI (blue). (M) Quantification of Tuj1-positive neurons in the dorsal root ganglia showed no difference between the WT and KO samples. Scale bars: (A,B) and (E,F) 1000 µm, (K,L) 100 µm.
Article Snippet: The primary antibodies against NCAM1 (ab5032, 1:500, Merck), IGSF3 (AF4788, 1:750, R&D Systems),Tuj1 (AB18207, 1:1000, Abcam) and αSMA-CY343 (C6198, 1:300, Merck) were applied for 48 h at 4 °C followed by washes with 0.3% Triton-X-100 in PBS for the whole day and the secondary antibody donkey anti-rabbit Alexa Fluor 647 (A32795, 1:400, Thermo Fisher Scientific) or donkey anti-goat Alexa Fluor 488 (705–546-147, 1:400, Jackson ImmunoResearch) overnight at 4°C followed by washes 14 Vol:. (1234567890) Scientific Reports | (2023) 13:17162 | https://doi.org/10.1038/s41598-023-44093-8 for 3–4 h using 0.3% Triton-X-100 in PBS.
Techniques: Migration, Cell Culture, Isolation, Staining