Anti th

Lung paraffin sections were slided into 3μm-thick, dewaxed in xylol 5min for twice, and then rehydrated in 100% ethanol 5min for twice, 75% ethanol 5min and tap water for 5min. The sections were washed by phosphate-buffered saline (PBS) 3min for twice. 3% Hydrogen peroxide and methanol mixed together and the slides were incubated in order to inactive endogenous peroxidases. Then the slides were put into 0.1% pepsin for antigen retrieval. Next procedure was blocked in protein block solution and incubated with primary antibodies: TGF-β1 (Abcam, USA, 1:400), anti-GAP43 (Abcam, USA, 1:1000), anti-TH (Abcam, USA, 1:300), Collagen I (Bioss, China, 1:300), anti-CollagenIII (Bioss, China, 1:300) at 37°C for 2h, then overnight at 4°C. The slides were washed by PBS, then followed by incubation with biotinylated rabbit anti-IgG (ZSGB-BIO, China) and stained with DAB kit (ZSGB-BIO, China) and hematoxylin. The software (Image-pro plus 6.0, Meida Cybernetics LP) was applied to the analysis of the lung section. We measured the density of positive cells in total lung section.

After treated with MPP⁺ or OPA, collected SH-SY5Y cells were lysed by RIPA (Beyotime, China). The lysate was incubated on ice for 30 min and then centrifuged at 12,000 g for 5 min at 4°C. Meanwhile, the taken-out striatum of PD mice model was homogenized by adding an appropriate amount of RIPA according to the quality, and then, it was centrifuged at 1,000 g for 15 min at 4°C. Both supernatants were collected, and the concentration of total protein in supernatants was quantified using BCA protein assay kit (Beyotime, China). Next, the protein was subjected to 12% polyacrylamide gels and transferred onto a polyvinylidene fluoride (PVDF) (Millipore, United States). After blocking for 1 h with 5% non-fat milk at room temperature, the membrane was incubated with primary antibodies, including anti-Bax, anti-Bcl-2, anti-cleaved-caspase3, anti-β-actin (Cell Signalling Technology, United States), and anti-TH (Abcam, United Kingdom) overnight at 4°C. Following being washed by PBS/Tween-20 for three times, the membranes were incubated in the secondary antibody (1:2,000) at room temperature for 1 h. After being rinsed, bands were visualized using western blotting detection system (Bio-rad ChemiDoc Touch, United States) and quantified with NIH ImageJ software (National Institutes of Health, Bethesda, United States). β-actin was named as the internal control.