Several days after transplantation, head and tail parts were removed from recipients. we developed a technological platform for transplanting cells into the lesion site of SCI-treated adult zebrafish and defined the optimum number of successfully transplanted cells into recipients, as 300, and those GFP-(+) donor cells could enhance recipients spinal cord regeneration. Thus, we provided a practical methodology for studying cell transplantation therapy in neuronal regeneration of zebrafish after SCI. were cultured indoors21, and their developmental stages were identified according to standard procedures described by Westerfield22. All embryos were cultured in embryo medium (EM) (140 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, and 4.2 mM NaHCO3 at pH 7.2) for 1 day, and the medium was replaced by fresh EM containing 0.003% 1-phenyl-2-thiourea (Sigma, St. Louis, MO, USA). Fixation and Bone Decalcification of Adult Zebrafish We followed the procedures described by Kroehne et al.23 with some modifications. After we collected the adult zebrafish, they were anesthetized with 100 mg/l Tricaine (the ethylester of 3-aminobenzoic acid; Sigma) and both head and tail were removed. We then fixed trunk samples in a 4% paraformaldehyde (pH 7.2 to 7.4; Sigma) at 25C for 3 days, followed by treating with 20% of decalcification stock solution (2.5M EDTA, Thermo, Waltham, USA; final concentration of 0.5M EDTA, pH 7.8) at 25C for 7 days, washing three times with phosphate-buffered saline (PBS) (pH 7.4), keeping samples at 4C. Frozen Section of Adult Zebrafish We followed the procedures described by Zeng et al.20, except we used a 25-m thick slice. The green fluorescence signal shown on each section was detected by primary polyclonal antibody against GFP (Abcam, Cambridge, UK) and observed by an A1 confocal laser-scanning microscope (Nikon, Nagoya, Japan). The green fluorescence signal shown on each frozen section was detected by primary polyclonal antibody against GFP (Abcam, Cambridge, UK) at 1:150 dilution, anti-phospho-histone 3 (PH3) (Millipore, Bedford, MA, USA) at 1:200, and anti-HuCD (Fausett et al., 2006?) (early neuronal marker; Invitrogen, Carlsbad, CA, USA) at 1:25024 . The secondary antibody was either goat anti-rabbit or anti-mouse Cy3-conjugated fluorescence (Millipore) at 1:500. Confocal Microscopy and Imaging Processes The fluorescent signal shown on embryos was observed by Batimastat sodium salt a Zeiss confocal microscope (LSM 780, Rabbit Polyclonal to HBAP1 Carl Zeiss AG) and an A1 confocal laser-scanning microscope (Nikon). The image processing software with NIS-Elements Confocal and Zeiss LSM 780 (Carl Zeiss AG) Confocal was used for image capture. Hypoxic Exposure of Zebrafish Embryos from Transgenic Line embryos to hypoxia to induce GFP-expressing cells (Fig. 1A and B). Second, we used the FACS sorter to isolate GFP-(+) cells, which served as donor cells (Fig. 1C). Meanwhile, we administered mechanical SCI to adult WT zebrafish (Fig. 1D). The GFP-(+) cells were then transplanted into the lesion site of SCI-treated adult zebrafish. Open in a separate window Physique 1. GFP-(+) cells were transplanted into the spinal cord lesion site of SCI-treated adult zebrafish. (A) Schematic illustration of the procedures for treating and transplanting cells to zebrafish transgenic line larvae. Therefore, it is reasonable for us Batimastat sodium salt to hypothesize that this transplanted GFP-expressing cells at the lesion site of SCI-treated adult zebrafish are not only able to undergo proliferation but also able to differentiate into functional neurons. To confirm this hypothesis, we collected SCI-treated WT adult zebrafish after transplantation for 5 and 7 days and performed frozen cross sections to detect the proliferation and differentiation of cells transplanted into recipients using proliferation marker PH3 and early neuron marker HuCD, respectively. Thereafter, we examined whether the transplanted GFP-expressing donor cells could also express PH3 and HuCD Batimastat sodium salt markers labeled with red fluorescent protein (RFP) at 5 and 7 dpt. The results exhibited that this GFP-expressing cells transplanted into recipients did exhibit yellow fluorescence signal, which was due to the colocalization of two signals, GFP and RFP (Fig. 4). Besides, 53.33% 8.50% GFP-expressing cells could express HuCD at 7 dpt. The line of evidence.