Alexa fluor 594 nm conjugated secondary antibodies

Immunostaining was performed as previously described [17 (link),21 (link),42 (link),43 (link),75 (link)]. Briefly, stem cells on PBCE*, PBCE, BGD10 and BDG30 squares, and on TCP and GC were rinsed twice with PBS, fixed in 4% paraformaldehyde for 20 min, washed twice with PBS, and permeabilized and blocked (PBS + 3% FBS, 0.3% Triton X-100) for 1 h at RT. Samples were incubated with phalloidin (Alexa-fluor-488 phalloidin, Invitrogen) for 20 min at RT, or overnight at 4 °C with the primary human antibodies: anti-MAP2 (Abnova, Taipei, Taiwan), anti-GFAP, anti-NG2 (Millipore, Billerica, MA, USA), anti-TJU1, anti-nestin, and anti-βTubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). In the latter cases, after 3 washing with PBS, the staining with Alexa-Fluor 488-nm or Alexa-Fluor 594-nm conjugated secondary antibodies (Invitrogen) for 1 h at room temperature was performed. After being washed with PBS, samples were mounted and nuclei were counterstained with Vectashield with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Images were acquired using fluorescence microscopy (Eclipse-TE2000-S, Nikon, Tokyo, Japan) equipped with the F-ViewII FireWire camera (Soft Imaging System, Olympus, Münster, Germany). Images were elaborated as described below. Interference of PBCE*, PBCE, BDG10 and BDG30 squares without cells to a fluorescence microscope (Eclipse-TE2000-S) was evaluated.

Immunofluorescence experiments, cells were rinsed twice with PBS, fixed in 4% paraformaldehyde for 30 min and, after PBS washing, cells were permeabilized, blocked (PBS + 10% FBS, and 0.1% Triton X-100) for 1 h at RT (room temperature), and incubated with phalloidin (Alexa-fluor-488 phalloidin, Invitrogen, Grand Island, NY, USA), for 20 min then further incubated overnight at 4 °C with other primary antibodies: anti-α-tubulin, anti-osteocalcin (Santa-Cruz Biotechnology, Santa Cruz, CA, USA) and anti-vinculin (clone hVIN-1, Sigma-Aldrich, St Louis, MO, USA). Finally, after washing with PBS and staining with Alexa-Fluor-594 nm conjugated secondary antibodies (Invitrogen) for 1 h at RT, cover slips were mounted and nuclei were counterstained with Vectashield with DAPI (Vector Laboratories Inc., Burlingame, CA, USA).
Images were acquired with fluorescence microscopy (Eclipse-TE2000-S, Nikon) using the F-ViewII FireWire^TM camera (Olympus Soft Imaging System, Olympus, Münster, Germany). Image casting was made by Adobe Photoshop CS5 program.