Dapi h 1200

After AAV viral injection for 4 weeks, mice were anesthetized with pelltobarbitalum natricum and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde (PFA 4%) dissolved in 0.1 mol phosphate buffered saline (PBS 0.1 mol). Brains were extracted and post fixed in PFA 4% for 24 h. Brains were transferred to 15% sucrose for 48–72 h and then to 30% sucrose for 48–72 h before slicing 20 μm coronal frozen sections of the entire hippocampus. Sections were mounted on slides and cover slipped using mounting media with DAPI (H-1200, Vector) to label cell nuclei and stored at 4 °C.

Paraffin embedded tissue sections were dewaxed utilising 4 changes of xylene (X/0250, Thermo Fisher Scientific, Loughborough, United Kingdom) for 5 min each and rehydrated using descending ethanol series [100, 90 and 75% (v/v) for 2 min each]. Following dewaxing and rehydration, tissue sections of control and decellularised pulp tissues were stained using standard histological procedures with haematoxylin and eosin (H&E; RRSP60-D, Thermo Fisher Scientific), 4′,6-diamidino-2-phenylindole (DAPI, H-1200, Vector Laboratories Ltd, Peterborough, United Kingdom), alcian blue (RRSK400, TCS Biosciences Ltd, Buckingham, United Kingdom), and picrosirius red (24901, Polysciences, Inc. Northampton, United Kingdom). Following staining; the slides were dehydrated using ascending ethanol series [75% for 5 min and 3 changes of 100% (v/v) for 20 s], cleared in four successive changes of xylene for 5 min each and then mounted with synthetic resin media (DPX mountant, REA212, Solmedia Ltd, Shrewsbury, United Kingdom). All slides were viewed using either normal Köhler illumination, polarised light or under reflected light and a DAPI filter (λ_ex = 365 nm/λ_em = 445/450 nm) with images captured digitally using an AxioCam and AxioVision or Zen software (ZEISS, Cambridge, United Kingdom) and qualitatively assessed.

Cells from BALF and blood were subjected to cytospinning, fixed, permeabilized, and stained with Ly6G (LS-C36561, 1:100; LifeSpan Biosciences) or MPO (ab9525, 1:100, abcam) antibodies as primary antibodies. The following day, goat anti-rabbit IgG (H&L) Alexa Fluor® 488 (1:400, ab150077; Abcam) or goat anti-rabbit IgG (H&L) Cy5® (ab6564, 1:400; Abcam) was incubated as a secondary antibody at 37 °C for 2 h. Cell nuclei were counterstained with DAPI (H-1200; Vector Laboratories, CA). Images of the cells were obtained under a Fluoview confocal microscope (FV10i; Olympus).

Five-micron sections of formalin–fixed, paraffin-embedded lung tissue were stained with hematoxylin and eosin by standard procedures at the University of Colorado Histology Core to assess vessel wall thickness. Immunohistochemistry analysis for α-smooth muscle actin, 4-hydroxynonenal, interlukin-6 (IL-6) was performed per the manufacturer’s instructions. Antigen retrieval was performed on serial lung sections and then incubated with antibodies against smooth muscle actin (1:500 abcam, ab5694, Cambridge, MA), 4HNE (1:500 abcam, ab46545 Cambridge, MA) and IL-6 (1:50, Santa Cruz, sc-1265). Lung sections were incubated with a fluorescent secondary antibody (either Alexa Fluor 488 goat anti-rabbit IgG (1:300 or 1:800; A11008) or Alexa Fluor 555 (1:500; A21422) Invitrogen, Carlsbad, CA). Primary antibodies were incubated at 4°C overnight, and Secondary antibody was incubated for 1 hour at room temperature. Slides were then mounted with Vectashield mounting medium for fluorescence with dapi (H-1200) from Vector and coversliped. Pulmonary vessels (outside diameter ~100 μm) were assessed for the presence of smooth muscle actin, 4HNE and IL-6 protein expression on a Nikon Eclipse Ti-E inverted epi-fluorescent microscope (Nikon Instruments, Tokyo Japan).

We performed immunofluorescence labeling to determine ubiquitin and α‐syn expression levels in SH‐SY5Y cells. Cells were fixed in 4% paraformaldehyde and then washed three times with phosphate‐buffered saline (PBS). The cells were then blocked with 10% BSA containing 0.05% Triton X‐100 at room temperature for 30 min. Next, the cells were incubated with anti‐Ubiquitin (1:200, Abcam, Cat. #ab7780), anti‐α‐syn (1:200, CST, Cat. #4179), and anti‐Parkin antibody (1:200, Abcam, Cat. #ab77924) at 4°C overnight. Then, the cells were incubated with secondary antibody for 1 hr at room temperature. DAPI (H‐1200; Vector) was used to stain the nuclei. Images were obtained via a fluorescence microscope or a N‐SIM Nikon Super‐Resolution Microscope (Nikon).