Nestin antibody

Primary hepatocytes were fixed in 4% paraformaldehyde and made into smear. Immunofluorescence was performed as previous described [9 (link)]. Albumin antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD31 antibody (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Laminin antibody (1:100, Abcam, Cambridge, UK), and Nestin antibody (1:50, Chemicon, Billerica, MA, USA) were used. FITC-conjugated donkey anti-goat antibody or Cy3-conjugated goat anti-rabbit antibody was used as secondary antibodies (1:100, Jackson Immuno-Research, West Grove, PA). At last, nuclei were stained with DAPI.

The NPCs were washed with 3% bovine serum albumin (BSA) in PBS twice, then the NPCs were permeabilized with 0.2% Triton X-100 in PBS at 4^˚C for 10 min, and blocked with 3% BSA at room temperature for 1 h. NPCs were further incubated with nestin antibody (1:200, Chemicon, Temecula, CA) overnight at 4^˚C, followed by staining with Alexa Fluor® 594 goat anti-mouse IgG (Invitrogen, Carlsbad, CA) for 1 h at room temperature and away from light. Finally, the cells were incubated with 4’, 6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) in a humidified dark chamber for 10 min, and then were analyzed in mounting medium under a fluorescence microscope.

hMSC characterization by flow cytometry was performed on cells before seeding on biomaterials to demonstrate their characteristics as defined by the ISCT criteria (Dominici et al., 2006 (link)). hMSC were stained with CD90-FITC, CD73-PE, CD105-PC7, CD45-APC-A750 antibodies (Beckman Coulter, Brea, California, United States), or corresponding isotype controls in matched concentration, 20 min at room temperature (RT) and in the dark. Cells were washed in PBS without Ca²⁺/Mg²⁺ (Thermo Fisher), fixed, and permeabilized (IntraPrep Permeabilization Kit, Beckman Coulter). Intracellular staining was performed by an indirect immunofluorescence method with an adapted dilution of Nestin antibody (Merck Millipore, Burlington, Massachusetts, United States), or its corresponding isotype control in matched concentration, and with an Alexa Fluor 647 goat anti-mouse IgG (H + L) secondary antibody (Thermo Fisher). After washing, cells were analyzed with a NAVIOS flow cytometer (Beckman Coulter) and data files were interpretated using Kaluza software (Beckman Coulter).