Ab243228

Mice were narcotized, blood was collected from the abdominal aorta, and EDTA anticoagulant was added. Blood lymphocytes of mice were isolated by blood lymphocyte separation kit, the second layer of milky white lymphocytes was carefully moved to another tube. After centrifugation, the collected lymphocytes were re-suspended with PBS, and antibodies of CD4 (Abcam, USA, ab183685, Rabbit monoclonal [EPR19514] to CD4), CD28 (Abcam, USA, ab243228, Rabbit monoclonal [EPR22076] to CD28), CTLA-4 (Abcam, USA, ab237712, Rabbit monoclonal [CAL49] to CTLA4), PD-1 (CST, USA, #84,651, Rabbit monoclonal [D7D5W] to -PD-1), and MHCII (Sigma, USA, SAB4700663, Rat monoclonal [M5/114] to - MHCII) were added respectively for flow cytometry .

Paraffin-embedded substantia nigra pars compacta (SNpc) brain tissue blocks were sectioned at 5 μm. The citric acid antigen repair buffer was placed in the microwave oven for antigen repair, and the sections were placed in 3% hydrogen peroxide solution to block endogenous peroxidase. 3% BSA was added for blocking at room temperature for 30 min. Then anti-CD4 (Abcam, USA, ab183685, Rabbit monoclonal [EPR19514] to CD4), anti-CD28 (Abcam, USA, ab243228, Rabbit monoclonal [EPR22076] to CD28), anti-CTLA-4 (Abcam, USA, ab237712, Rabbit monoclonal [CAL49] to CTLA4), anti-PD-1 (CST, USA, #84,651, Rabbit monoclonal [D7D5W] to -PD-1) and anti-MHCII (Sigma, USA, SAB4700663, Rat monoclonal [M5/114] to - MHCII) antibodies were separately added to examine the number of infiltrating T cells in the brain. The tissue sections were covered with the secondary antibody (Abcam, USA, ab150113/ab150077) and incubated at room temperature for 50 min. DBA kit was used for color development, hematoxylin was used to re-stain the nucleus. After gradient alcohol dehydration and sealing, the slices were observed under a microscope. At least 3 slices per mouse in the different groups (10 mice per group, n=10) were used in the analysis.