Alexa fluor 488 labelled donkey anti mouse

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.

The PFA-fixed tumour tissues were embedded in paraffin, cut to 5-μm-thick sections using a microtome, and transferred onto glass slides. After baking, the slides were processed through serial steps to deparaffinize in Tissue-Clear (Cat. No. 1466, Sakura) and rehydrate tissues in 99–95–70% ethanol. For cryosection staining, tissue slides were fixed with acetone (Cat. No. 32201, Sigma). Rehydrated tissues were washed in PBS, boiled in a unmasking solution and subsequently blocked with 4% serum before incubation overnight at 4 °C with primary antibodies against F4/80 (Rabbit, Cat. No. NBP2-12506, Novus Biologicals), Iba1 (rabbit, Cat. No. 019-19741, WAKO), PDGFRα (Rat, Cat. No. 14-1401-82, eBioscience), PDGFRβ (Rat, Cat. No. 14-1402-81, eBioscience), NG2 (Rabbit, Cat. No. AB5320, Millipore) or αSMA (mouse, Cat. No. M0851, DAKO). The tissue slides were incubated for 30 min with fluorescent-labelled secondary antibodies including: an Alexa Fluor 555-labelled goat anti-rat antibody (Cat. No. A21434, Invitrogen); an Alexa Fluor 488-labelled donkey anti-mouse (Cat. No. A21202, Invitrogen); and an Alexa Fluor 488-labelled donkey anti-rabbit (Cat. No. A21206, Invitrogen) antibody. The slides were thoroughly washed and mounted with Vectashield containing DAPI. The positive signals were analysed under a fluorescence microscope (Zeiss LSM510 Confocal, or Nikon C1 Confocal microscope).