Goat anti mouse ngn1

Western blot analysis was performed as described by M He, et al [29 (link)]. After ELF-EMF exposure, protein samples were harvested and lysed in RIPA buffer (Thermo Scientific, USA) containing a cocktail of protease inhibitors (Roche, USA). The protein concentration was determined using a BCA protein assay kit (Takara, Japan). Equal amounts of protein were load for SDS-PAGE. After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad, USA). Then, the membranes were blocked and incubated with various primary antibodies at 4°C overnight. Mouse anti-mouse TRPC1 (1:1000, Santa Cruz, USA), rabbit anti-mouse NeuroD (1:500, Santa Cruz, USA), goat anti-mouse Ngn1 (1:500, Santa Cruz, USA), and mouse anti-mouse GAPDH (1:5000, Abcam, USA) were used as primary antibodies. The next day, the membranes were washed in Tris-buffered saline-Tween 20 to remove unbound primary antibodies, incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime, China), and finally examined using an electrochemiluminescence system (Thermo Fisher Scientific, USA). The bands were imaged and analyzed using a ChemiDoc XRS + System with Image Lab Software (Bio-Rad, USA).

eNSCs (1.0×10⁵ cells in 1 ml) were plated onto poly-L-lysine-coated glass coverslips and were exposed to ELF-EMF for 3 days in differentiation medium. Then the cells were immediately fixed with 4°C 4% paraformaldehyde for 20 min (for neuron staining) or fixed after another 3 days of culture (for astrocytes staining). Immunocytochemistry was carried out as previously described [26 (link)]. The primary antibodies of mouse anti-mouse Tuj1 (1:100, R&D, USA) and rabbit anti-mouse GFAP (1:200, Beijing Zhongshan, Beijing, China) were used to stain the neurons and astrocytes, respectively. The other applied primary antibodies were as follow: rabbit anti-mouse TRPC1 (1:100, Abcam, USA), rabbit anti-mouse NeuroD (1:100, Santa Cruz, USA), goat anti-mouse Ngn1 (1:50, Santa Cruz, USA). Alexa Fluor 488-labelled donkey anti-mouse, Alexa Fluor 555-labelled donkey anti-rabbit and Alexa Fluor 647-labelled donkey anti-goat secondary antibodies (1:200, Invitrogen, USA) were used for visualization. Cell nuclei were stained with Hoechst33342 (5 μg/ml). Tuj1⁺ and GFAP⁺ cells were counted in four different fields of each coverslip using a 63×objective under a Leica TCS SP5 confocal fluorescence microscope. More than 1000 cells on 12 coverslips from five independent experiments were counted.