Ab5968

For investigation of surface and intracellular antigen expression profiles, TSs were transferred to cover slides, fixed with 2 % paraformaldehyde for 7 min, and then treated with a 3:1 ratio of methanol and acetic acid for 3 min. The cells were then washed and permeabilized by incubating with 0.1 % Triton X-100 for 10 min. After blocking with 1 % bovine serum albumin (BSA; Amresco, Solon, OH, USA) for 1 h, cells were incubated with primary antibodies for 2 h at room temperature. The following antibodies were used: rabbit anti-CD133 (1:250, ab19898; Abcam [Dawinbio Inc], Hanam, Korea), rabbit anti-nestin (1:250, ab5968; Abcam). Primary antibodies against CD133 and nestin were detected with goat anti-rabbit IgG conjugated with Alexa Fluor 555 (1:2000; Invitrogen), which is spectrally similar to Cy3. The cells were mounted with Vectashield H-1200 mounting media containing 4′6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA) to counterstain nuclei. Phosphate-buffered saline (PBS; Dawinbio Inc, Hanam, Korea) was used for all washing steps, and antibody diluent reagent solution (Invitrogen) was used to dilute antibodies. As a negative control, only the secondary antibody was used. A fluorescence microscope (1 × 71; Olympus Korea, Seoul, Korea) and DP Controller software (Olympus Korea) were used for observing and photographing the cells.

Tooth cultures were fixed overnight at 4°C in 4% paraformaldehyde (SigmaAldrich). Subsequently, they were cryoprotected overnight at 4°C in 30% sucrose and then embedded in OCT (Tissue-Tek) and stored at −80°C until use. Frozen sections (15 μm) were obtained using a cryostat (Shandon) and post-fixed in 4% paraformaldehyde for 10 min. Some sections were stained with hematoxylin-eosin or permeabilized using 1% Triton X-100 (SigmaAldrich) in PBS (SigmaAldrich) and blocked with 10% fetal bovine serum for 1 h at room temperature. These samples were incubated overnight at 4°C with the following antibodies:mouse anti-bromodeoxyuridine-fluorescein monoclonal antibody 1:200 (Roche 11202693001), rabbit anti-cleaved Caspase3 1:200 (Asp175; Cell Signaling) anti-Nestin 1:100 (ab5968; Abcam) and anti-Amelogenin 1:200 (ab59705 Abcam) antibodies. The secondary antibody used was Alexa Fluor 488-conjugated anti-rabbit antiserum (dilution 1:500). Cell nuclei were stained with DAPI (4′,6-Diamidino-2-phenylindole, SigmaAldrich) 0.5 μg/ml. Immunofluorescences were performed by triplicate and images were obtained using an Olympus FluoView FV500 confocal microscope or a Zeiss Axioskop fluorescence microscope with Nikon DS-Qi1Mc and Nikon NIS-Elements software, respectively.