Thy1.1 monoclonal antibody

Retinal ganglion cells grown on cover slips were fixed in 4% paraformaldehyde as previously described7 (link)8 9 (link). Briefly, cells were blocked in 5% BSA for 1 h at room temperature, followed by overnight incubation at 4 °C with the following primary antibodies: GPR91 polyclonal antibody (1:200, Novus Biologicals, Littleton, CO, USA), Thy1.1 monoclonal antibody (1:200), c-Fos monoclonal antibody (1:200, Cell Signaling Technology, Boston, MA, USA), C/EBP β monoclonal antibody (1:200, Novus Biologicals, Littleton, CO, USA), and C/EBP δ (1:200, Santa Cruz, CA). The cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies, phycoerythrin PE-conjugated anti-mouse secondary antibodies or PE-conjugated anti-rabbit secondary antibodies (1:200, Invitrogen, Carlsbad, CA) for 1 h at room temperature. Fluorescence imaging and analyses were performed using a fluorescence microscope (Leica, Wetzlar, Germany).

RGC-5 cells were grown on coverslips and fixed with 4% paraformaldehyde and blocked with blocking solution (1% BSA, 22.52 mg/ml glycine in PBST) for 1 hour at room temperature. The blocking buffer was removed, and the coverslips were washed three times with 1X PBST, before the addition of the primary antibody (Thy1.1 monoclonal antibody; Cell Signaling, Brn3a monoclonal antibody; Abcam, 1/500 dilution) with 1% BSA in PBST. The incubation was performed overnight at 4 °C. Coverslips were washed three times, 1:500 of secondary antibody (Alexa Fluor 488; Invitrogen-Molecular Probes) was added, and the coverslips were incubated for 1 hour in the dark. After the incubation, the coverslips were washed again three times with 1X PBST. The coverslips were mounted on glass slides (Prolong Gold antifade reagent with DAPI; Invitrogen). The cells were viewed with a fluorescence microscope (TE2000-U; NIKON, JAPAN).