Mouse monoclonal anti tuj1

Immunocytochemical staining was conducted according to our previously described protocol 31 (link). The following primary antibodies were used for staining: mouse monoclonal anti-Tuj1 (1:100; Millipore, Billerica, MA, USA), rabbit polyclonal anti-MAP2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:200; Millipore), mouse monoclonal anti-glutamic acid decarboxylase (GAD67) (1:200; Abcam, Cambridge, UK), rabbit polyclonal anti-glutamate transporter (GluT) (1:200; Abcam), and rabbit polyclonal anti-neurofilament 200 (NF200) (1:200; Sigma). The following secondary antibodies were used: Alexa Fluor-488 goat anti-mouse IgG (1:500) and Alexa Fluor-488 donkey anti-rabbit IgG (1:500) (Invitrogen). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma). The fluorescently stained signals were detected under a confocal microscope (LSM 700, Carl Zeiss, Jena, Germany). Neurite formation and cell body length were quantified from Tuj1- or MAP2-stained cell images as described in our previous study 24 (link). Focal adhesion staining (vinculin) was performed using the FAK100 kit (Millipore).

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature, washed 3 times with DPBS, and then incubated in DPBS containing 0.5% Triton X-100, 1% bovine serum albumin (BSA) fraction V (both from Sigma-Aldrich), and 10% fetal bovine serum for 10 min at room temperature. The cells were briefly rinsed with DPBS and incubated with primary antibodies overnight at 4°C or for 1 h at room temperature. The following primary antibodies were used: mouse monoclonal anti-SSEA1 (DSHB, 1:200), mouse monoclonal anti-Tuj1 (Millipore, 1:200), mouse monoclonal anti-SMA (R&D Systems, 1:200) and mouse monoclonal anti- AFP (R&D Systems, 1:200). The cells were then washed with 0.5% BSA in PBS and incubated with secondary antibodies (anti-mouse IgG or anti-mouse IgM; both from R&D Systems, 1:200) for 1 h at room temperature. Nuclei were detected by Hoechst 33342 (Fluka) staining.