A total of 12 rats were randomly divided into 2 groups (6 rats/group): The control (saline) and model (6-OHDA). On post-surgery day 28, a total of 11 rats (1 rat from the model group that did not reach the modeling standard was excluded from the experiment) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally. The cardiac aorta injection, blood washing and brain tissue fixation were performed using normal cold saline and 4% paraformaldehyde solution. Frozen sections (20-µm-thick) were subjected to antigen retrieval under high pressure using a heated citrate buffer. Immunohistochemical staining was performed using Histostain-Plus kits (cat. no. SP-0022, BIOSS) according to the manufacturer's protocol. Tissues were incubated in the presence of primary antibodies against TH (1:250; cat. no. AB152, EMD Millipore), GPX4 (1:250; cat. no. ab125066, Abcam) and FTH1 (1:250; cat. no. ab183781, Abcam) at 4°C overnight. After washing, the sections were incubated with secondary antibody (cat. no. ab6721; horseradish peroxidase-conjugated goat anti-rabbit; 1:5,000; Abcam). Then, diaminobenzidine (DAB) (cat. no. C02-04001; BIOSS) was added, and controlled color development was performed under a light microscope (Olympus, Japan) for strictly controlled amounts of time. Tissues were counter-stained with hematoxylin (cat. no. BA4097, BaSO Biotech) for 10 sec at room temperature, dehydrated gradually with ethanol and coverslipped. Proteins of interest were quantified and analyzed using ImageJ software (version 1.52a; National Institutes of Health). The threshold feature in the menu of ImageJ software was used to count immune cells. Each immune cell was quantified using three IHC images, and the upper, middle, lower, left and right parts of each IHC image were selected.

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