In general, DRG NSCs were washed in 0.1 M phosphate-buffered saline (PBS) and treated with 3% H2O2 in PBS to quench the endogenous peroxidase activity. For VGluT2 immunostaining, cells were first incubated in 1% Triton X-100 (Tx-100) overnight at 4°C. For in vitro immunostaining, nonspecific binding was blocked using 4% normal serum from the secondary antibody host species, plus 0.1% Tx-100 in PBS (blocking buffer). Primary antibodies (detailed in Table 1) were incubated overnight at room temperature in the corresponding blocking buffer above. For immunofluorescent staining, DRG NSCs were washed with PBS and incubated with an Alexa 350, 488, or 635 fluorophor-conjugated secondary antibody (against the primary antibody species) at room temperature for 90 min. Immunoperoxidase staining utilized a biotinylated secondary antibody and avidin-biotin peroxidase kit (ABC Elite, Vector Laboratories, Burlingame, CA). Peroxidase activity was revealed by incubation in 0.1% diaminobenzidine tetra-hydrochloride (DAB) in 0.05 M Tris-HCl buffer, followed by 0.003% H2O2, allowing visualization of the chromagen. Slides were counterstained with methyl green.
