Bs1258

Retinal tissues were carefully isolated, fixed in 4% paraformaldehyde overnight, embedded in paraffin and prepared into 4-μm sections. The sections were stained with H&E and observed under a microscope (Nikon, Tokyo, Japan). For immunohistochemistry staining, the paraffin-embedded retinal tissue sections were dewaxed and exposed to 3% hydrogen peroxide for 20 min to inhibit endogenous peroxidase activity. After socking in boiled citrate buffer (pH 6.0, 10 mM) for 30 min to repair antigen activity, the sections were blocked with 5% bovine serum albumin (BSA) for 1 h and incubated with the primary antibody at 4 °C overnight. Subsequently, the sections were washed with PBS, incubated with secondary antibody for 1 h at 37 °C, visualized using diaminobenzidine, counterstained with hematoxylin and observed under a microscope (Nikon, Tokyo, Japan). The primary antibodies included cleaved caspase-3 (1:100, CST, 9661), NRF2 (1:100, Bioworld, BS1258), PTEN (1:100, Bioworld, BS1305), p-AKT (1:100, CST, 4060) and NEDD4 (1:100, Proteintech, 21698-1-AP).

The brain tissues of the nigrostriatum were homogenized, and the total proteins of the samples were extracted by radioimmunoprecipitation assay lysis buffer (P0013B, Beyotime, Shanghai, China) and centrifuged at 12,000 revolutions/min for 20 min, and the supernatant was taken. The protein concentration was measured with BCA kit (P0010S, Beyotime, Shanghai, China) and adjusted to the same level. After heat denaturation, the protein samples were added to 12% SDS-PAGE gel for electrophoresis, transferred to polyvinylidene fluoride membrane, and sealed with 5% skimmed milk powder solution for 2 h. Subsequently, the membrane was incubated overnight in a primary antibody of synuclein-α (1:1,000, BS3429, Bioworld, MN, USA), TH (1:1,000, BS1432, Bioworld, MN, USA), Keap1 (1:1,000, BS6783, Bioworld, MN, USA), Nrf2 (1:1,000, BS1258, Bioworld, MN, USA), SOD1 (1:1,000, BS6057, Bioworld, MN, USA), GPx1 (1:1,000, BS61511, Bioworld, MN, USA), and β-actin (1:1,000, AP0060, Bioworld, Minnesota, USA) at 4℃ and then transferred to diluent containing horseradish peroxidase-labeled secondary antibody (1:5,000, A0208, Beyotime, Shanghai, China) for further incubation. The membrane was added with chemiluminescent solution, and the gray values of each protein were recorded and analyzed by Image J software. β-actin was used as the internal reference.

WB was performed according to our previous study method [17 (link), 35 (link)]. We used the following primary antibodies to perform the WB analyses: rabbit monoclonal anti-NF-κB p65 (D14E12) XP® antibody (1:1000, #8242, Cell Signaling Technology); rabbit polyclonal anti-Nrf2 (L593) antibody (1:500, BS1258, Bioworld); mouse monoclonal anti-Cryopyrin (NLRP3) (6F12) antibody (1:1000, sc-134306, Santa Cruz Biotechnology); rabbit polyclonal anti-PYCARD (ASC) antibody (1:500, A1170, Abclonal); goat polyclonal anti-caspase-1 p20 (M-19) antibody(1:1000, sc-1218, Santa Cruz Biotechnology); rabbit polyclonal anti-IL-1β (H-153) antibody (1: 1000, sc-7884, Santa Cruz Biotechnology); and rabbit polyclonal anti-IL-18 (H-173) antibody (1:1000, sc-7954, Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1000, Cell Signaling Technology) was used as an internal reference. Blot bands were quantified via densitometry with ImageJ software (National Institutes of Health, Baltimore, MD, USA), and protein levels were expressed as the ratio of values of the detected protein bands to that of GAPDH bands.