Goat anti mouse immunoglobulin g igg

Cells were fixed with 4% paraformaldehyde (PFA; USB Products, Cleveland, OH, USA) for 30 min and washed with phosphate-buffered saline (PBS). Fixed cells were blocked with 5% normal goat serum (Millipore, Temecula, CA, USA) and 0.2% Triton X-100 (Amresco) in PBS for 40 min. The cells were then incubated for more than 1.5 h with primary antibodies: anti-βIII tubulin antibody (TuJ1, mouse monoclonal antibody, 1:1000; Sigma-Aldrich), anti-GFAP (rabbit polyclonal antibody, 1:1000; Dako, Glostrup, Denmark), and anti-GalC (mouse monoclonal antibody, 1:500; Millipore). After rinsing with PBS, the cells were incubated for 1 h with secondary antibodies conjugated to Alexa Fluor 488 (goat anti-mouse immunoglobulin G [IgG], 1:1000; Invitrogen), or Cy3 (goat anti-rabbit IgG, 1:1000; Jackson ImmunoResearch, West Grove, PA, USA). 4′-6-diamidino-2-phenylindole (DAPI, 1:10000 in PBS; Sigma-Aldrich) was added for 5 min to stain the nuclei. The images were obtained using an inverse fluorescence microscope (DMIL; Leica, Wetzlar, Germany). To avoid a bias in measurement, the photos were randomly taken, and TuJ1-, GFAP-, GalC- or DAPI-positive cells were counted. The number of TuJ1-, GFAP- or GalC- positive cells was divided by that of DAPI-positive cells to obtain the percentage.

Immunocytochemical examination was performed as previously described [37 (link)]. Cell cultures were fixed with 4% paraformaldehyde (PFA; USB Products, OH, USA) for 30 min and washed with phosphate-buffered saline (PBS). Fixed cells were blocked with 5% normal goat serum (Millipore, CA, USA) and 0.2% Triton X-100 (Amresco, OH, USA) in PBS for 30 min and incubated with antibodies against βIII Tubulin (TuJ1, mouse monoclonal antibody, 1:1000; Sigma-Aldrich), glial fibrillary acidic protein (GFAP, rabbit polyclonal antibody, 1:1000; Dako, Copenhagen, Denmark), S100β (mouse monoclonal antibody, 1:500; Sigma-Aldrich), and Ki67 (rabbit monoclonal antibody, 1:400; Thermo Scientific, CA, USA) for 1 h. After PBS rinses, the cells were incubated for 30 min with secondary antibodies conjugated to Alexa Fluor 488 (goat anti-mouse immunoglobulin G [IgG], 1:1000; Invitrogen), Alexa Fluor 546 (goat anti-mouse IgG₁, 1:1000; Invitrogen) or Cy3 (goat anti-rabbit IgG, 1:1000; Jackson ImmunoResearch, PA, USA) followed by 5 min in 4′,6-diamidino-2-phenylindole (DAPI, 1:10000 in PBS; Sigma-Aldrich) to stain the nuclei. Images were obtained using an inverse fluorescence microscope (DMIL; Leica, Hesse, Germany). TuJ1-, GFAP-, S100β-, or Ki67-positive cells were counted and normalized to total DAPI-positive cell numbers.