Ab128848

Total protein was extracted from tissues and cells using RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with 1% phosphorylase inhibitor and 1% protease inhibitor, with the concentration determined using a BCA protein assay kit (GBCBIO Technologies Inc., Guangzhou, China). Next, the protein was separated with 10% SDS‐PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). The membrane was blocked with TBST with 5% BSA at room temperature and probed overnight at 4°C with primary rabbit antibodies to TREM2 (1:1000, #61788, Cell Signaling Technologies, Beverly, MA), TGF‐β1 (1:800, ab215715, Abcam), Smad2/3 (1:500, ab236030, Abcam), p‐Smad2/3 (1:1500, ab272332, Abcam), DAT (1:200, ab128848, Abcam), TH (1:5000, ab137869, Abcam), and GAPDH (ab9485, 1:2500, Abcam) and then with secondary antibody goat anti‐rabbit IgG (1:10,000, ab97051, Abcam) at room temperature. ECL reagent was used to visualize the results by Image Quant LAS 4000C Gel Imager (GE Healthcare, Waukesha, WI). ImageJ software (National Institutes of Health) was applied for band intensity quantification, with GAPDH as an internal reference.

Nigral tissue sections were subjected to microwave‐stimulated antigen retrieval with 1 mM Tris‐EDTA (pH 8.0) and treated with 3% H₂O₂ at room temperature for 10 min. Next, sections were immunostained with primary antibodies to TH (1:500, ab137869, Abcam Inc., Cambridge, UK) and DAT (1:500, ab128848, Abcam) at 4°C overnight. The next day, the sections were incubated with polymer enhancer (PV‐9000, ZSGB‐Bio, Beijing, China) at room temperature for 20 min, and then with enzyme‐labeled anti‐mouse/rabbit polymer (PV‐9000, ZSGB‐Bio) at room temperature for 30 min. Thereafter, the sections were developed with DAB for 5 min, counterstained with hematoxylin, hydrolyzed, blued, dehydrated, cleared and mounted. Finally, the sections were observed under an inverted microscope (CX41, Olympus).