Pa5 34974

Rats were anesthetized and transcardially perfused with ice cold 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were postfixed in 4% PFA and then cryoprotected in 30% sucrose solution. 40 μm coronal slices were sectioned on the microtome and then subsequently were stored at 4°C in PBS with 0.01% sodium azide until processing. Sections were washed in PBS and then incubated for 20 minutes at room temperature in 3% normal horse serum. A solution of primary antibodies (1:500 rabbit anti-mCherry, Invitrogen #PA5–34974 or Abcam, Cambridge, UK, Cat #167453; and 1:500 guinea pig anti-cFos, Synaptic Systems, Göttingen, GER, Cat#226 308) was diluted in 3% normal goat serum and 0.05% Triton-X solution and then applied overnight at room temperature. Sections were then washed in PBS and a solution of fluorophore-conjugated secondary antibodies (1:300 horse anti-rabbit 594, Vector, Olean, NY, Cat#DI-1094 and 1:200 goat-anti guinea pig 488, Abcam, Cambridge, UK, Cat#150185 or Invitrogen, Waltham, MA, Cat#A11073) were applied at room temperature for 30 minutes. Finally, sections were washed in PBS and mounted on slides with DAPI Fluoromount-G (Southern Biotech, Birmingham, AL, Cat#0100–20). To quantify activity-dependent tagging, LHb sections were imaged for mCherry and Fos and overlap manually counted.

STHdh cells and primary cortical neurons were grown on poly-D-Lysine-coated coverslips. Cells were fixed with 4% paraformaldehyde for 15–20 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 (in phosphate buffered saline, PBS), blocked with PBS containing 10% FBS and 0.05% Triton X-100 for 1h at room temperature. Primary antibodies were incubated overnight at 4°C in blocking solution with the following dilutions: HTT (Merck-Millipore #MAB2166, 1:100), PRMT2 (Abcam #ab66763, 1:100), PRMT6 (Abcam #ab47244, 1:100), mCherry (Life Technologies #PA5-34974, 1:600), MAP2 (Abcam #ab11267, 1:500 or Merck-Millipore #AB15452, 1:100), GFP (Merck-Millipore #MAB2510, 1:1000). The next day, after three washes with PBS, Alexa Fluor conjugated secondary antibodies (1:1000) were incubated for 1h at room temperature in the dark. The coverslips were then washed three times with PBS, incubated with Hoechst (Sigma #B2261) diluted in PBS for 10 min and mounted on glass slides with ProLong Diamond Antifade Mountant (Life technologies #P36961). Primary neurons and STHdh slides were imaged with a 63x oil-immersion objective using the Zeiss Axio Observer Z1 inverted microscope or an inverted confocal microscope (LSM 710, Zeiss) coupled to an Airyscan detector, respectively.