Gtx30920

Figure 3B illustrates the flowchart used for the animal transfection experiment. Animals were sacrificed following 1-MHz focused US and completion of pPrestin-MB treatment (1, 2, 7, 14, and 21 days; 60 s, 120 s, and 240 s; n=4 in each condition) and were perfused with 4% paraformaldehyde. The frozen brains were sliced into coronal sections. The activity and location of gene transfection were identified using the intracerebral expression of green fluorescence protein (Venus) via microscope. The cellular nuclei were labelled by DAPI (GTX30920, GeneTex, Inc., TX, USA). The contralateral brain without gene transfection was used for comparison. The expression of Prestin was estimated by calculating the intensity of green fluorescence within ROI.

After 3 months of WGE treatment, mice were sacrificed at the age of 11.5 months. The substantia nigra and striatum were dissected out. The substantia nigra was subjected to immunostaining, as described previously (Lin et al., 2020 (link)). Anti-tyrosine hydroxylase (TH) (Millipore, AB152, 1:200) and anti-ionized calcium-binding adapter molecule 1 (Iba-1) (GeneTex, GTX100042, 1:200) were used as primary antibodies for 24 hr at 4°C. Secondary antibodies were DyLight 488 goat anti-rabbit 1:300 and Alexa Fluor 546 goat anti-rabbit at 1:200 (25°C for 1 hr). Mounting medium with DAPI (GeneTex, GTX30920) was used as a counterstain.

Mice were subjected to cardiac perfusion with 50 mL PBS and 50 mL of 4% paraformaldehyde (PFA) in PBS. Brain tissues were collected and fixed again with 4% PFA in PBS overnight. After dehydrating with 10%, 20%, and 30% sucrose in PBS, the brains were embedded in optimal cutting temperature (OCT) compound (4583, Sakula Finetek, Torrance, CA, USA) for frozen slices. After permeabilization with 0.2% Triton X-100 in PBS for 15 mins at room temperature (RT), brain sections were then incubated with 3% goat serum (ab7481, Abcam) and 3% BSA in 0.2% Triton X-100 in PBS for blocking. Sections were labeled with primary antibodies including TH (1:500, rabbit, ab137869, Abcam), ATPB (1:200, mouse, ab14730, Abcam), DARPP-32 (1:500, rabbit, GTX133350, Genetex), and Ctip2 (1:200, rat, ab18465, Abcam) at 4°C overnight, followed by incubation with fluorescent dye-conjugated secondary antibodies for 2 h at 37°C. Finally, the slices were mounted with Fluoroshield™, which contains the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) (GTX30920; Genetex) for fluorescence detection. As a negative control, the primary antibody was omitted and observers were blinded. All the fluorescence was imaged in the sections using the THUNDER Imaging Systems (Dmi8 S, Leica, Wetzlar, Germany) by an observer who was blinded to all three groups.