Mouse α-βIII tubulin antibody was prepared in house (20 (link)), Rabbit anti- βIII (T2200) was purchased from Sigma Aldrich, Alexa Fluor 488 cross-linked Goat anti-Mouse (A11029) and anti-Rabbit (A11034) antibodies were purchased from Life Technologies. Primary antibodies α-GSK3β (9832), α-cJun (3270), α-Cofilin-1 (5175), α-Erk1/2 (9107), α-NF-κB (6956), α-pSer9 GSK3β (5558), α-pSer73 c-Jun (2315), α-pThr202/Tyr204 Erk1/2 (4370), and α-pSer276 NF-κB (3031) were purchased from Cell Signaling; α-pSer3 Cofilin-1 (sc-271923) was purchased from Santa Cruz Biotechnology. Secondary antibodies α-mouse-[IR800] (926-32210) and α-mouse-[IR680] (926-32220) were purchased from LiCor, while α-rabbit-[IR800] (611-732-127) and α-rabbit-[IR680] (611-130-002) were purchased from Rockland. Poly-D-Lysine (P7886-500MG) and sterile dimethyl sulfoxide (DMSO) (D2650) were purchased from Sigma Aldrich. Hippocampal tissue was incubated in Hibernate E from BrainBits, supplemented with NeuroCult® SM1 (05711) from StemCell Technologies. Neurons were cultured in NbActive4® media from BrainBits. Accell siRNA SMARTpools were purchased from Thermo Scientific. All other reagents were purchased from Life Technologies. In vitro kinase profiling was done at NanoSyn.
Nbactive4 media
Manufactured by Transnetyx
NbActive4® media is a cell culture medium designed for the maintenance and growth of cells. It provides the necessary nutrients and growth factors to support cell viability and proliferation.
Automatically generated - may contain errors
2 protocols using nbactive4 media
1
Neuron Culture and Antibody Protocol
2
Immunostaining of Dissociated Ganglion Neurons
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Bilateral NGs resected from mice were immersed in hibernateA media (BrainBits). Each ganglion was cut into five pieces, placed in HibernateA minus Ca2+ media (BrainBits) containing 1 mg/ml collagenase/dispase (Roche Diagnostics) and incubated for 90 min at 37 °C. Neurons were dispersed by gentle titration through pipettes and they were washed three times with fresh NbActive4 media (BrainBits) and plated onto poly-D-lysine/laminin (BD Bioscience)-coated coverslips, followed by incubation (5% CO2 in air at 37 °C) for 24 h. Cultures were rinsed in 10 mM phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB). Ganglion neurons were incubated with the first primary antibodies; anti-GLP-1R or anti-GHSR at 4 °C overnight, and then with the second primary antibodies; anti-GHSR or anti-Pan Neuronal Marker (Supplementary Table S1 ) at 4 °C overnight. They were then reacted with corresponding Alexa Fluor 488- or 594-conjugated secondary IgG (Supplementary Table S1 ) at RT for 1 h. Neurons were observed under a fluorescence microscope (Olympus).
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