A11120

Immunostaining of brain sections was used to validate AAV-directed gene targeting or expression (mice were euthanized 2 to 3 weeks after AAV administration) and to image DA neuron neurite density in the NAc. Brain tissues were collected after perfusion with PBS and 4% paraformaldehyde (PFA). PFA-fixed brain tissues were cryostat-sectioned at 40 μm thickness. Sections were permeabilized with 0.5% Triton X-100 for 30 min and then blocked in 5% normal goat serum for 1 hour at room temperature. Free-floating coronal sections were immunostained with anti-Becn2 (Millipore, ABC253), anti-GFP (Invitrogen, A11120), and anti-TH (Millipore, AB152 and MAB318) antibodies and then with Alexa Fluor or Alexa Fluor Plus 488–conjugated secondary antibody (Invitrogen, A-11008, A-11001, and A32723) and Alexa Fluor 594–conjugated secondary antibody (Invitrogen, A-11012 and A-11005). Slides were mounted using ProLong Diamond Antifade Mountant (Invitrogen, P36961). Fluorescence images were acquired using a Nikon CSU-W1 spinning disk confocal microscope. The images were analyzed using Nikon’s NIS-Elements.

Ovaries were dissected in ice-cold PBS solution. Dissected ovaries were fixed in 4% paraformaldehyde in 1x PBS for 15 minutes at room temperature. Primary antibodies were added and then incubated for overnight at 4°C. Antibodies used in this study are rabbit anti-GFP (Invitrogen #A11122, 1:1000), mouse anti-GFP (Invitrogen #A11120, 1:1000), rabbit anti-H3K4Me2 (Millipore #07-030, 1:1000), rabbit anti-H3K27Ac (Abcam #ab4729, 1:1000), rabbit anti-H3K27Me3 (Cell signaling #9733, 1:1000), mouse anti-Lsd1 (1:2500,²⁵ (link) mouse anti-Hnt (DSHB #1G9, 1:20) and mouse anti-Cut (DSHB #2B10, 1:25). Secondary antibodies used are goat anti-rabbit 488 (Invitrogen #A11008, 1:500), goat anti-mouse 488 (Invitrogen #A11001, 1:500), goat anti-mouse 568 (Invitrogen #A11004, 1:500) and goat anti-rabbit 568 (Invitrogen #A11011, 1:500). Stained ovaries were mounted in mounting medium with DAPI (EMS#17989-20) on glass slides. Images were taken on Zeiss Axio Imager 2/Apotome.2 microscope and processed with ImageJ software. As previously described, the number of follicle cells was quantified by taking pictures of stage 10 egg chambers and counting the number of nuclei (based on DAPI signals) per side using ImageJ software.²⁵ (link)