Fig. 1. Custom injection apparatus assembly.

The injection apparatus consists of a manual hydraulic manipulator that advances a plunger through the bore of a pulled glass pipette. Three of the four custom parts (see Supplementary File 1 for fabrication information) are mounted on the vertical rod that comes with the stereotax and hold both the micromanipulator and the pipette; the fourth is mounted on the moveable shaft of the manipulator and holds the plunger that moves within the bore of the pipette to expel the virus. For best results, adhere to the positioning of the components shown here. The vertical metal rod extends approximately 10.5 cm below the metal clamp that holds the vertical rod to the stereotax arm, and the bottom of the pipette-clamping block is attached to the vertical rod (with a 4–40 screw) and flush with the end of it. The two symmetrical micromanipulator-clamping blocks are clamped (with two brass 10–32 screws) to both the vertical rod and the micromanipulator; the blocks should be flush against the metal clamp, and the end of the micromanipulator shaft is ~1.8 cm below the bottom edge of the clamping blocks. The plunger-clamping block is attached (with a 4–40 screw) to the piston of the micromanipulator, with the grooves of the pipette-clamping block and those of the plunger-clamping block aligned so that the plunger is in line with the pipette. The two 4–40 thumbscrews that pin the pipette and its plunger into the grooves in their respective blocks have nylon washers to provide grip and prevent breakage of the glass. While the exact positioning of the custom parts on the stereotax rod is not critical, the positions shown here work well, and major deviation from them could result in the inability to raise the pipette high enough to allow the stereotax arm to be swung out of the way when inserting and removing the mouse or, conversely, the inability to lower the pipette to the desired depth in the brain.