Once the depth of anesthesia was confirmed by the lack of reflex to a tail pinch or light touching of the cornea, animals were perfused with 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde and 0.1% glutaraldehyde at a rate of 50 ml/min during the first 3 minutes, followed by perfusion with the identical solution but at a rate of 20 ml/min for the succeeding 7 minutes. Brains were left in the skull for 1 hour post perfusion to allow in situ fixation to continue.
