Fujiki, R., et al. Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP transgenic mice. J. Vis. Exp. (2019)
The video demonstrates the isolation of oculomotor, trochlear, and spinal motor neurons from a transgenic mouse embryo. The embryo expresses motor neurons labeled with a green fluorescent protein (GFP) for easy visualization and identification. The motor neurons are surgically isolated under a fluorescence microscope and maintained in a culture medium.
The authors have nothing to disclose.
B27 Supplement (50X), serum free | Thermo Fisher Scientific | 17504-044 | |
BD FACSAria llu SORP Flow Cytometer | BD Bioscience | – | This has 4 laser system equipped with 405, 488, 594, and 640 nm lasers. |
BD Falcon 70μm Nylon Cell Strainers | CORNING | 352350 | For filtering the dissociating cells before FACS. |
BD Falcon Round Bottom Test Tubes With Snap Cap | CORNING | 352054 | |
BDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-207 | |
Cell Culture microplate, 96 well, PS, F-bottom (Chimney Well) | Greiner Bio-One International | 655090 | We tried multiple 96-well dishes and this was the best one for culture and analyses after ICC |
CNTF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-272 | |
4′,6-diamidino-2-phenylinodole (DAPI) | Thermo Fisher Scientific | D1306 | |
Dimethyl sulfoxide | Sigma-Aldrich | D2650 | DMSO |
Dumont #5 Forceps Inox Tip Size .05 x .01 mm Biologie Tips | Roboz Surgical Instrument | RS-5015 | |
Forskolin | Thermo Fisher Scientific | BP25205 | |
GDNF Human | ProSpec-Tany TechnoGene, Ltd. | CYT-305 | |
GlutaMAX supplement | Thermo Fisher Scientific | 35050-061 | |
Hanks' Balanced Salt Solution (HBSS) | Thermo Fisher Scientific | 14175-095 | |
Hibernate E | BrainBits | HE | |
Hibernate E low fluorescence | BrainBits | HELF | Fluorescence which hinders observation of embryo's GFP expressions should be low. |
Horse serum, heat inactivated, New Zealand origin | Thermo Fisher Scientific | 26050-070 | |
IBMX | Tocris Cookson | 2845 | Isobutylmethylxanthine |
Laminin | Thermo Fisher Scientific | 23017-015 | |
Leibovitz's L15 medium | Thermo Fisher Scientific | 11415064 | |
2-Mercaptoethanol | Sigma-Aldrich | M6250 | |
Micro Dissecting Scissors | Roboz Surgical Instrument | RS-5913 | |
Micro Knife 4.75" 1.7 x 27 mm blade | Roboz Surgical Instrument | RS-6272 | |
Moria Mini Perforated Spoon | Fine Science Tools | 10370-19 | |
Olympus 1.7ml Microtubes, Clear | Genesee Scientific | 22-281 | These are the tubes that we described "1.7 mL microcentrifuge tubes" in the context. |
Papain Dissociation System | Worthington Biochemical Corp | LK003150 | Papain solution and alubumin- ovomucoid inhibitor solution are prepared from this kit. |
Penicillin-streptomycin (10,000 U/ ml) | Thermo Fisher Scientific | 15140-122 | |
Phosphate buffered saline (PBS) | Thermo Fisher Scientific | 10010-023 | |
Poly D-lysin (PDL) | MilliporeSigma | A-003-E | |
SMZ18 and SMZ1500 zoom stereomicroscopes with DS-Ri1 camera | Nikon | – | Dissection was performed and images of dissected embryos and tissues are captured under these fluorescence microscopes. |
Sylgard 170 Black Silicone Encapsulant – A+B 0.9 Kg kit | Dow Corning | 1696157 | We make dissection dishes using this kit. |
TC treated Dishes, 100 x 20 mm | Genesee Scientific | 25-202 | We make dissection dishes using this dish. |
Thum Dressing Forceps 4.5" Serrated 2.2 mm Tip Width | Roboz Surgical Instrument | RS-8100 | |
Zeiss LSM 700 series laser scanning confocal microscope and Zen Software | Carl Zeiss | – | Confocal image of the embryo was captured with these equipments |