As previously described (Riessland et al., 2019 (link)), 3 weeks post injection mice were anesthetized with pentobarbital and transcardially perfused using PBS (pH 7.4), followed by 4% paraformaldehyde in PBS. Brains were postfixed in 4% paraformaldehyde in PBS at room temperature for 1 h and then cryopreserved using a gradient of 5%, 15%, and 30% sucrose. The brains were embedded in Neg‐50 (Thermo Scientific), frozen and stored at −80°C, and cut into 14‐μm‐thick coronal sections using a Microm cryostat and thaw‐mounted onto Superfrost Plus microscope slides (Thermo Scientific). For staining of TH, brain sections were washed in PBS and permeabilized with 0.2% Triton X‐100 in PBS, followed by blocking with 2% donkey serum and 0.1% fish gelatin in 0.2% Triton X‐100 in PBS. Sections were then incubated with rabbit polyclonal anti‐TH antibody (Millipore, #AB152) at a concentration of 1:250 overnight. The next day, slides were washed with PBS, incubated with Alexa Fluor 633 goat anti–rabbit IgG (Life Technologies, #A21070) for 2 h at room temperature, and then after another washing step were mounted with ProLong Diamond Antifade containing DAPI (Life Technologies, #P36962), coverslipped, and stored in the dark until imaging.
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