Mouse anti nestin antibody

To check for the expression of typical SKP and SC markers, immunocytochemistry was achieved. After fixed in 4 % paraformaldehyde for 10 min, cells were blocked with 5 % normal goat serum and permeabilized with 0.1 % Triton-X for 30 min. Primary antibody was incubated overnight at 4 °C, followed by a secondary antibody incubation for 2 h at room temperature. Where Hoechst was used, 1:5000 Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was added for incubating 5 min. The following primary antibodies were used at the stated dilutions: mouse anti-nestin antibody (1:200, Millipore, Temecula, CA, USA), rabbit anti-Sca-1 (1:200, Merck Millipore, Darmstadt, Germany), rabbit anti-versican, fibronectin, vimentin, collagen I, collagen IV (1:200, Abcam, Cambridge, MA, USA), chicken anti-P0 (1:500, Aves Labs, Davis, CA, USA), mouse anti-S100β and anti-laminin (1:500, Sigma Aldrich, St Louis, MO, USA), rabbit anti-GFAP (1:500, Dako, Tokyo, Japan), mouse anti-p75^NTR (1:500, Chemicon, Temecula, CA, USA), mouse anti-NF200 (1:400, Abcam, Cambridge, MA,USA), and rabbit anti-MBP (1:500, Millipore, Billerica, MA, USA). The following secondary antibodies were used at the stated dilutions: Alexa 555 goat anti-chicken, Alexa 555 goat anti-rabbit, Alexa 488 goat anti-mouse, Alexa 555 goat anti-mouse, (all 1:1000, all Invitrogen, Carlsbad, CA, USA).