Monoclonal anti β galactosidase antibody

Embryonic neurons from the embryos of puc^E69-lacZ/TM6B were plated on concanavalin A–coated, 25-mm photoetched coverslips (#72265-25; Electron Microscopy Sciences, Hatfield, PA) for >48 h. Neurons were first injured by a glass needle (see Embryonic neuronal axotomy procedure for more details) with the position information recorded according to the grids. After axotomy, neurons were cultured overnight (>16 h) to allow the expression of the reporter. Then neurons were washed three times in 1× phosphate-buffered saline (PBS) before fixation in 4% formaldehyde for 10 min at room temperature and then washed three times in 1× PBS and washed and blocked in wash solution (0.1% Triton X-100, 1% bovine serum albumin in Tris-buffered saline) for 10 min three times. Then they were incubated with the primary antibody, a monoclonal anti–β-galactosidase antibody (1:250; Cat# Z3781; Promega, Madison, WI), for 45 min at room temperature in a moisture chamber, washed and blocked in wash solution 10 min three times, incubated with secondary antibody, goat anti-mouse tetramethylrhodamine isothiocyanate (1:200; Jackson Immuno­Research, West Grove, PA) for 45 min to 1 h in the dark at room temperature, and washed and blocked in wash solution 10 min three times. Cells were covered with 1× PBS and imaged directly according to the grid position information.