Anti tuj1 antibody

For PSD95 (Cat #SC6926, Santa Cruz), vGluT1 (Cat #75-066, Antibodies Inc.) and GluR1 (Cat #SC7608, Santa Cruz) immunostaining, fixed D14 primary hippocampal neurons were permeabilized with 0.2% Triton and blocked with 20% NHS for 1 h at room temperature. The cells were then immune-labelled in PSB containing 2% NHS overnight at 4 °C. Biotin-conjugated antibodies were used to enhance the staining. Anti-Tuj1 antibody (Neuron-specific class III beta-tubulin; Cat #845502, BioLegend) was used to identify the cells. Three to five dendritic segments per cell were used to quantify the number of puncta using the plugin ITCN of ImageJ. Data are presented as an average of 3 independent experiments.

The retinas were fixed in 4% PFA for 30 min and dissected into the petal shape as the whole flat-mount. The retinas were blocked with 5% BSA for 60 min, incubated with anti-TUJ1 antibody (Dilution ratio: 1:200, Biolegend, 801201, USA) overnight at 4°C. Then, the retinas were washed with PBS buffer and incubated with the Alexa Fluor 594-conjugated goat anti-mouse IgG (Dilution ratio: 1:500, Invitrogen, USA) for 3 h at room temperature. TUJ1-positive cells were counted using the ImagePro Plus 6.0 (Media Cybernetics, USA) software. RGC survival rate was evaluated by dividing the average number of TUJ1-positive cells in the injured retinas by that in normal retinas.