Mouse anti nestin

Neurospheres that adhered to poly L-lysine-coated slides were used for studies of surface markers. Primary antibodies used included mouse anti-nestin (1:250, Abcam) and rabbit anti-CD133 (1:5, Miltenyi Biotec GmbH). Secondary antibodies were anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 (Molecular Probes, Eugene, OR, USA). Images were captured by fluorescence microscope.
Neurospheres were dissociated into single cell suspensions, and then incubated with PE-conjugated nestin (1:100; Miltenyi Biotech GmbH) and APC-conjugated CD133 (1:100; Miltenyi Biotech GmbH) antibody. Mouse anti-TfR primary antibody (1:200, Invitrogen) and PE-conjugated mouse IgG (1:100, Invitrogen) were used. Labeled cells were analyzed by flow cytometry (Beckton Dickinson FACScan; BD Biosciences). Cells were collected and lysed for western blotting analysis by standard procedures. Primary antibodies of anti-TfR (1:1000) and anti-β-actin (Sigma,USA) were used.

1 × 10⁴ cell/cm² of hSCAPs were seeded on 24 well plate (Corning) and cultured for 2 days. The attached cells were fixed with 3.7% formaldehyde (Sigma) for 20 mins at room temperature, and fixed cells were washed with PBS. The cells were permeabilized with 0.2% Triton X-100 (Sigma) treatment for 20 mins, and, after brief washing with PBS, were treated with 4% bovine serum albumin (BSA: Sigma) to block non-specific binding of antibodies at 4°C for overnight. After blocking, the cells were incubated with primary antibodies such as 1:200-diluted mouse-anti-nestin (Abcam) antibody, 1:200-diluted mouse-anti-Stro-1 (Abcam) antibody, and 1:200-diluted mouse-anti-CD44 (Abcam), and 1:200-diluted mouse-anti-CD133 (Abcam) at 4°C for overnight. After reaction with primary antibodies, the cells were washed gently with PBS three times, and then incubated with secondary antibodies such as 1:2000-diluted alexa 488 (goat anti-mouse IgG, Invitrogen), 1:2000-diluted alexa 596 (goat anti-mouse IgG, Invitrogen) for 1 hr at room temperature under dark condition. Finally, after brief washing with PBS three times, the cells were counter-stained with DAPI, and mounted. The stained cells were observed under an inverted fluorescence microscope (Olympus, IX-72).

For FACS analysis, the neurospheres were dissociated into single cell by accutase digestion. Single cell suspensions were then stained with appropriate antibodies as follows: mouse anti-nestin (1 : 50, Abcam), rat anti-CXCR4 (1 : 50, BioLegend), and rabbit anti-βIII tubulin (1 : 10, Epitomics). All flow cytometric analysis was performed using FACSCAria II (BD Biosciences) and results were analyzed by FACSDiva software (BD Biosciences).