Mouse monoclonal anti nestin antibody

The stored frozen sections were rinsed in 0.1 M PBS (3 times for 5 min each time) at
first, permeabilized with 0.5% Triton X-100 (Beyotime, Shanghai, China) and incubated with
3% bovine serum albumin (BSA, Boster, Wuhan, China) at room temperature. The sections were
incubated at 4°C overnight with the following primary antibodies: rabbit polyclonal
anti-Ki-67 antibody (1:200, Abcam, Cambridge, MA, USA), rabbit polyclonal
anti-doublecortin (DCX) antibody (1:100, Abcam), rabbit polyclonal anti-glial fibrillary
acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-Nestin antibody
(1:100, Abcam). Afterward, the sections were incubated in the corresponding secondary
antibodies, goat anti-rabbit IgG (Alexa Fluor 555, Invitrogen, Carlsbad, CA, USA) or goat
anti-mouse IgG (Alexa Fluor 488, Invitrogen), at 37°C for 1 h, and
4′,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei at room temperature for 10
min. Slides were made for observation and storage. Immunofluorescence was examined by
confocal microscopy (LSM800, ZEISS, Oberkochen, Germany), and images were obtained using
an LSM Image Examiner. The numbers of Nestin-, DCX-, GFAP-, and Ki-67-positive cells were
counted using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and incubated in blocking solution (1% bovine serum albumin in phosphate buffered saline (PBS)) for 30 min before incubation with mouse monoclonal anti-nestin antibody (1:200; Abcam, Cambridge, UK) at 4°C overnight. Cells were then incubated with fluorescein isothiocyanate-conjugated goat anti-mouse secondary antibodies (1:200; Jackson Immunoresearch, UK) at 37°C for 1 h, and mounted in Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK).

The cultured cells were seeded onto six-well plates, and their differentiation potency was characterized and tested using immunofluorescence staining analysis. To fix the cells, 4% paraformaldehyde (PFA) was used. Mouse monoclonal anti-nestin antibody (1:500, Abcam, Boston, MA, USA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-β-tubulin III antibody (1:500, Abcam) were used for NPCs identification. D4′, 6′-diamidion-2′-phenylindole (DAPI, 1:1000, Abcam) was used to counterstain the nuclei. To detect CypD and Complex V (mitochondrial marker), anti-CypD (1:200, ab110324, Abcam) and anti-complex V (1:200, 45, 9000, Invitrogen, Carlsbad, CA, USA) antibodies were used. After fixing the cells for 20 min, they were placed in 0.1% Triton X-100 for 30 min, followed by a blocking buffer (10% goat serum) for 1 h. The cells were then incubated with primary antibodies overnight at 4 °C. Samples were incubated in secondary antibodies [fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG] for 2 h, followed by DAPI incubation for 5 min at room temperature. The slides were rinsed with DPBS, and the images were captured using a laser confocal microscope (TSC SP2, Leica, Manheim, Germany).