Anti rabbit igg h l antibody

Rat adenohypophysis tissues were fixed in 4% paraformaldehyde (MA0192, Meilunbio, Dalian, China) for 48 h and then made into paraffin sections. After dewaxing and rehydration, the sections were antigenically repaired using EDTA antigen retrieval solution (P0084, Beyotime, Shanghai, China). The samples were sequentially incubated in blocking solution (incubated for 30 min at room temperature), primary antibody (FTO antibody 1:2000 dilution, FOXP2 antibody 1:200 dilution, overnight at 4 °C), and secondary antibody (1:500 dilution, incubated for 1 h at room temperature away from light). The antibodies used were as follows: FTO antibody (ab280081, Abcam, UK); FOXP2 antibody (20,529–1-AP, Proteintech, USA); and anti-rabbit IgG (H + L) antibody (5220–0336, SeraCare, USA). Both antibody incubations were followed by washing three times with PBS (5 min at room temperature). The nucleus was restained with hematoxylin staining solution (C0107, Beyotime, Shanghai, China) after development using a DAB horseradish peroxidase color development kit (P0203, Beyotime, Shanghai, China). The sections were dehydrated and sealed. Images were visualized and collected using an Olympus fluorescence microscope. Three randomly selected images from the immunohistochemistry results were analyzed for positive reaction areas using ImageJ for statistical analysis.

Antibodies against the following proteins were used in this study: TH (abcam, ab137869; 1:5000), Trx-1 (Proteintech, 14999-1-AP; 1:2000), α-syn (abcam, ab212184; 1:2000), PINK1 (Santa Cruz Biotechnology, sc-517353; 1:500), Parkin (Santa Cruz Biotechnology, sc-32282; 1;500), LC3I/II (Cell Signaling Technology, #4108; 1:1000), p62 (Proteintech, 66184-1-Ig; 1:2000), cathepsin B (Proteintech, 12216-1-AP; 1:2000) cathepsin D (Santa Cruz Biotechnology, sc-377299; 1:500), LAMP2 (Proteintech, 66301-1-Ig; 1:2000), β-actin (Proteintech, 81115-1-RR; 1;10000), Anti-Mouse IgG (H + L) Antibody, (SeraCare, 5450-0011; 1:10000), Anti-Rabbit IgG (H + L) Antibody (SeraCare, 5450-0010; 1:10000). Fluorophore-conjugated secondary antibodies were as follows: Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (Thermo Fisher, A-11036; 1:1000). Other reagents included MPTP-HCl (Sigma-Aldrich-Corporation, 23007-85-4), MPP⁺ (Sigma-Aldrich-Corporation, 36913-39-0), Rapamycin (MedChemExpress, HY-10219), Chloroquine phosphate (MedChemExpress, HY-17589), Antifading Mounting Medium with DAPI, (Solarbio, S2110).

Total proteins were extracted using RIPA buffer (Beijing Zoman Biotechnology Co., Ltd, China) supplemented with the protease inhibitor cocktail (Beijing Zoman Biotechnology Co., Ltd) according to the manufacturer's instructions. Proteins (20 μg) were separated by 10% SDS-PAGE (Biotides, China) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% milk for two hours and incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with secondary antibodies at 37°C for one hour. Finally, proteins were visualized by using enzyme-linked chemiluminescence detection kit (ECL) and images were captured by a chemiluminescence imaging system (Azure Biosystems C300). The pixels from he western blot membranes were quantified by ImageJ software (NIH). Experiments were repeated three times.
NCM Universal Antibody Diluent was purchased from New Cell Molecular Biotech Co., Ltd. (China). Anti-mouse IgG antibody (H+L) (1:10000) and anti-rabbit IgG (H+L) antibody (1:10000) were purchased from Seracare (USA). Anti-beta actin (1:10000), anti-N-cadherin (1:1000), and anti-E-cadherin (1:1000) antibodies were purchased from GeneTex (USA). Anti-MMP-9 (1:1000) and anti-vimentin (1:5000) antibodies were purchased from Proteintech (China).