Rabbit α ha tag

For immunohistochemistry, mice were anesthetized and transcardially perfused using 25 mL PBS followed by 25 mL 4% paraformaldehyde (PFA). Brains were collected and post fixed for 24 hours in 4% PFA and transferred to a 30% sucrose for at least 48 hours. Brains were cryoprotected in Tissue-Tek optimal cooling temperature (OCT) compound and stored in a −80 freezer until use. 40 μm coronal sections were cut using a Leica cryostat. Brain slices were washed and permeabilized with PBST (0.4% Triton X-100 in PBS) over 15 minutes and incubated with blocking solution (5% normal goat serum in PBST) for 1 hour at room temperature. Slices were incubated in rabbit α-HA-tag (1:1000, Cell signaling: 3724S) overnight at 4°C. The following day slices were washed in PBST over 15 minutes and incubated in the AlexaFluor488-fluorescently-conjugated goat anti-rabbit secondary (1:250, Life Technologies) at RT for 1 hour. Finally, slices were mounted on slides with VECTASHIELD Antifade Mounting Media with DAPI (Vector Laboratories). Slices were imaged using Leica SPE Confocal Microscope maintained by the Iowa Neural Circuits and Behavioral Core.