Donkey anti chicken igg fluorescein isothiocyanate fitc

To identify the expression of cell protein markers, cell samples were fixed with 4% paraformaldehyde (PFA) (Beyotime, Shanghai, China) at room temperature (RT) for 15 min and then washed 3 times with phosphate buffer saline (PBS). Next, the cells were closed with the blocking solution (Beyotime) at RT for 1 h, then incubated with the primary antibody at 4 °C overnight, followed by incubation with the secondary antibody at RT in the dark for 2 h. Finally, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA), and the photomicrographs were taken by confocal microscope (SP5, Leica, Mannheim, Germany).
The primary antibodies used included mouse anti-S100β (1:500, Thermo Fisher Scientific, Waltham, MA, USA), chicken anti-glial fibrillary acidic protein (GFAP; 1:1,000, Abcam, Cambridge, MA, USA), mouse anti-β-tubulin3 (TUJ1) (1:400, bcam), rabbit anti-neurofilament 200 (NF200; 1:400, Sigma-Aldrich), and chicken anti-choline acetyltransferase (ChAT, 1:200, Abcam). The fluorescent-labeled secondary antibodies used included goat anti rabbit immunoglobulin G (IgG) cyanine 3 (Cy3) (1:400, Abcam), donkey anti chicken IgG Fluorescein isothiocyanate (FITC) (1:400, Abcam), goat anti mouse IgG 594 (1:400, Thermo Fisher Scientific), and goat anti mouse IgG FITC (1:400, Abcam).

To obtain MNs, on gestation day 13.5 a pregnant rat was isoflurane anesthetized, and sacrificed via cervical dislocation, and the fetal rats were removed from the uterus. The spinal cord of fetal rats soaked in Leibovitz's L-15 medium (Gibco, Carlsbad, CA, USA) was separated, then digested with 0.125% trypsin at 37 °C for 30 min. The purification of the MNs was performed in 15% OptiPrep gradient centrifugation solution (Sigma–Aldrich, St. Louis, MO, USA). Finally, the primary single cell suspension was obtained, and cultured on Poly-l-lysine (PLL) (Sigma–Aldrich) coated plates in DMEM medium (Corning) containing 1% penicillin/streptomycin and 10% FBS for 4 h. Next, culture medium changed to the ™Neurobasal medium (Gibco) containing 2% B27 (Gibco), 1% glutamine (Gibco) and 1% penicillin/streptomycin (Beyotime, Shanghai, China), and refreshed every other day.
In order to identify MNs, the primary antibody of chicken anti-choline acetyltransferase (ChAT) (1:200, Abcam) and mouse anti-β-tubulin III (TUJ1) (1:600, Abcam) were incubated with neurons at 4 °C overnight, followed by reaction with secondary antibody of goat anti mouse IgG-Cy3 (1:600, Jackson) and donkey anti chicken IgG-fluorescein isothiocyanate (FITC) (1:400, Abcam) at 25 °C for 2 h, then neurons were counterstained with Hoechst 33258. Images were captured by Confocal Microscope.

In our previous work, the primary rat SKP-SCs were obtained and cryopreserved at early passages [26 (link)]. Here, the cell resuscitation and expansion of SKP-SCs were performed for experimental application. The proliferating SKP-SCs were cultured in Schwann cell proliferation medium, namely DMEM/F12 medium (Corning, Manassas, VA, USA) containing 1% penicillin/streptomycin (Beyotime, Shanghai, China), 5 μm forskolin (Sigma–Aldrich, St Louis, MO, USA), 50 ng/mL heregulin-1β (R&D, Minneapolis, MN, USA), 2% N2 supplement (StemCell Technologies, Vancouver, BC, Canada), and 1–5% fetal bovine serum (FBS) (Sigma–Aldrich).
The identification of SKP-SCs were performed via immunocytochemistry staining with rabbit anti-S100β antibody (1:400, Invitrogen, Thermo Fisher Scientific, Rochester, MN, USA) and chicken-anti-glial fibrillary acidic protein (GFAP) antibody (1:1000, Abcam, Cambridge, UK) respectively at 4 °C overnight, followed by reaction with secondary antibody of goat anti-rabbit-IgM-Cy3 (1:400, Abcam) and donkey anti chicken IgG-fluorescein isothiocyanate (FITC) (1:400, Abcam) at 25 °C for 2 h, then cells were counterstained with Hoechst 33258. Images were captured by Confocal Microscope.