Brainbow3.0 rAAV (University of Pennsylvania, Penn Vector Core) injections were performed as previously described13 . Briefly, transgenic mice were anesthetized continuously with isoflurane and head-fixed to a stereotaxic apparatus. Surgery took place under sterile conditions with the animal lying on a heating pad. 2 μL AAV mix (7.5 × 1012 genome copy/mL) was injected at a rate of 0.2 μl/min through a 34-gauge injection needle into the brain (e.g., cortex, hippocampus), after which the needle was allowed to rest at the injection site for 5 min to allow viral diffusion. Animals expressed virus for 3–4 weeks, then were perfused (see “Mouse perfusion”).
Primary antibodies against Brainbow 3.0 fluorophores (chicken anti-GFP, guinea-pig anti-mKate2, rat anti-mTFP) were produced by the Cai lab. Slices were permeabilized and blocked with 1× PBS with 0.1% Triton X-100 and 2% normal donkey serum (PBT) for 30 minutes before antibody staining (all incubations at room temperature (RT)). Slices were incubated with primary antibodies for 3 days at 4°C in PBT, and then washed four times 30 minutes with PBT. Slices were incubated with secondary antibodies for 1 day at RT. Secondary antibodies used were: goat Anti-Chicken Alexa 488, goat Anti-Rat Alexa 546 (Life Technologies) and donkey Anti-Guinea Pig CF633 (Biotium), all at 10 μg/mL.
Primary antibodies against Brainbow 3.0 fluorophores (chicken anti-GFP, guinea-pig anti-mKate2, rat anti-mTFP) were produced by the Cai lab. Slices were permeabilized and blocked with 1× PBS with 0.1% Triton X-100 and 2% normal donkey serum (PBT) for 30 minutes before antibody staining (all incubations at room temperature (RT)). Slices were incubated with primary antibodies for 3 days at 4°C in PBT, and then washed four times 30 minutes with PBT. Slices were incubated with secondary antibodies for 1 day at RT. Secondary antibodies used were: goat Anti-Chicken Alexa 488, goat Anti-Rat Alexa 546 (Life Technologies) and donkey Anti-Guinea Pig CF633 (Biotium), all at 10 μg/mL.