Ab76442

Derivation of iPSC from MEFs was performed using STEMCCA approach (see Supplementary Methods for details). Colonies positive for alkaline phosphatase, Oct3/4, Zfp96, Nanog and ERas were expanded and banked for neural differentiation. The cultures were routinely checked and confirmed to be negative for mycoplasma.
Differentiation of iPSC into DA neurons was done using standard protocol42 (link) by first inducing formation of embryoid bodies in non-adherent conditions for 4 days in KSR media. Cells were then transferred to adherent plates and incubated in ITS media (DMEM/F12 (Gibco)+1 × ITS Supplement (Sigma I13146)+1 μg ml⁻¹ bovine fibronectin (Sigma F1141)) for 6–10 days to induce ectoderm formation. Cells were then expanded onto polyornithine-/fibronectin-coated coverslips in media containing N2 Max supplement, FGF2, FGF-8b, Shh-N and ascorbic acid for 5–7 days until cells reach confluency. Neural identity was confirmed by staining against βIII tubulin and nestin. Final differentiation into DA neurons was done by incubating the cells for 10 more days in minimal media (DMEM/F12 (Gibco)+1 × N2 Max+200 μM ascorbic acid (Sigma A4403)). Confirmation of DA neuron identity was done by staining the cells for TH (Abcam ab76442), Dopamine transporter (Abcam ab5990) and vesicular monoamine transporter 2 (Abcam 70808).

Dopamine primary neurons were prepared from the ventral midbrain of embryonic mice on day 13 of gestation. Ventral midbrain was isolated and dissected in ice‐cold Dulbecco’s medium + 0.2% BSA. Fragments were dissected into tiny pieces and collected in 2 ml vials. Then, pieces were washed three times in HBSS (Ca²⁺ and Mg²⁺ free) medium. Pieces were dissociated in HBSS containing 0.01% trypsin for 20 min at 37°C. Cells were dissociated by soft trituration in FBS medium containing 1 µg/ml of DNase I. After trituration, cells were washed in DA neuron culture medium (DMEM F12, N2 1X, 0.36% D‐(+)‐Glucose (wt/vol), primocin 100 µg/ml) and plated on 96‐well plates coated with poly‐L‐ornithine at a density of 5 × 10⁴ cells/well and let to adhere for 1 h. Then, cells were treated with netrin‐1 or GDNF. The treatment was renewed every 2 days for 4 days.
After treatment, primary neurons were fixed with 4% formaldehyde for 15 min and permeabilized with PBS solution containing and 0.2% Triton X‐100 for 20 min. Then, rinsed and incubated 1 h with a PBS blocking solution containing 2% NDS. Cells were next incubated with TH (Abcam, primary antibody ab76442) primary antibody overnight at 4°C. The next day, cells were rinsed and incubated for 1 h with donkey anti‐chicken fluorescent secondary antibody and then rinsed and kept at 4°C in PBS.

For histochemical analysis of post-mortem brain slices of normal subjects and PD patients, paraffin-embedded sections (6 µm) were stained with anti-CHCHD2 (66302-1-Ig, Proteintech, 1:500) or anti-TH (ab76442, Abcam, 1:500) antibodies. Quantitation of immunostaining was conducted using NIH image J software. The same image exposure times and threshold settings were used for sections from all groups. The immunohistochemical staining of brain tissues and quantification were performed in a double-blinded manner.

In brief, rabbit corneas were collected and fixed in Zamboni’s fixative solution for 4 h. After washing with PBS for three times, the corneas were blocked by PBS with 0.3% Triton X-100, 5% donkey serum overnight at 4°C, and subsequently incubated in the same incubation buffer with primary antibodies overnight at 4°C. After washing for five times, the corneas were incubated with fluorescein-conjugated secondary antibodies overnight at 4°C. The flat mounts were examined under a ZEISS confocal laser scanning microscope (ZEISS, LSM880, Germany). Mouse anti-beta III Tubulin (1:150, ab78078, Abcam, United States) and chicken anti-Tyrosine Hydroxylase (1:150, ab76442, Abcam, United States) were used as primary antibodies. Alexa Fluor 488- and 594-conjugated secondary antibodies (Life Technologies, United States) were used.

For fluorescent staining, primary antibody were as followed : Tyrosine Hydroxylase (TH, raised in Rabbit, 1:1000, AB152, Sigma-Aldrich), TH (raised in chicken, 1:1000, AB76442, Abcam), Choline acetyltransferase (ChAT, raised in goat, 1:1000, AB144P, Abcam), Alpha synuclein phosphorylated on serine 129 (pSer129, raised in Rabbit, 1:500, AB51253, Abcam), apolipoprotein E (ApoE, raised in goat, 1:500, 178479, Millipore).
Secondary antibody were as follow: 488-Anti Rabbit (raised in Donkey, 1:1000, A2206 , Jackson Immunoresearch); CY3-Anti Rabbit (raised in Goat, 1:1000, A11011, Thermofisher); CY5-Anti Rabbit (raised in Goat, 1:1000, A21244, Thermofisher); 488- Anti Chicken (raised in Goat, 1:1000, A11039, Thermofisher); CY3-Anti Chicken (raised in Donkey, 1:1000, A78951, Jackson Immunoresearch); CY5-Anti Chicken (raised in Donkey, 1:1000, A21449, Jackson Immunoresearch); CY3-Anti Goat (raised in Donkey, 1:1000, A11058, Jackson Immunoresearch); CY5-Anti Goat (raised in Donkey, 1:1000, A214447, Jackson Immunoresearch); 488- Anti Mouse (raised in Donkey, 1:1000, a21202, Thermofisher); CY3-Anti Mouse (raised in Donkey, 1:1000,715–175-150, Jackson Immunoresearch); CY5-Anti Mouse (raised in Donkey, 1:1000, 715–165-150, Jackson Immunoresearch); Biotin-conjugated goat Anti-Rabbit (1:500, B-6648, Sigma Aldrich).

Mice were culled by cervical dislocation. Brains were immediately dissected on ice before fixation in 4% paraformaldehyde at 4 °C with gentle shaking for 48–72 h. Tissue was washed in PBS, transferred to a 30% sucrose solution, incubated in an OCT:30% sucrose solution (1:1 ratio) for 1 h at RT, before being frozen in OCT over dry ice and stored for later use at − 80 ℃. After calibration in the Leica CM1800 cryostat (Leica Microsystems) chamber for 20 min, tissue was sectioned at − 22 ℃ to − 18 ℃ to generate 35 µm sections.
For immunofluorescence, sections were gently transferred into 10% Normal Goat Serum (NGS) blocking buffer for 1 h at RT, incubated in 0.2% phosphate-buffered serum with 0.1% TritonX (PBSTx) with 2% NGS with TH antibody (Abcam ab76442, 1:1000) overnight at 4 ℃ with shaking. The next day, sections were washed in PBS three times for 10 min, and then incubated for 1 h 45 min in 0.2% PBSTx with 2% NGS and secondary antibody (Invitrogen A-11039, Alexa 488, goat anti-chicken, 1:1000). Sections were washed in PBS twice for 10 min, incubated with DAPI for 30 min and washed again in PBS twice for 10 min. The sections were then floated in a larger basin with PBS and mounted on negatively charged slides, sealed with Vectashield, and left overnight in the dark before being examined.

Triple immunofluorescence staining was performed using 6-μm-thick, 4% paraformaldehyde-fixed, paraffin-embedded sections from the midbrain of clinically diagnosed and pathologically or genetically confirmed PD and control cases collected by the Department of Neurology, Juntendo University Hospital. Deparaffinized sections microwaved in Tris EDTA buffer, pH 9.0 (S2367, Agilent-DAKO, Santa Clara, CA) for 10 min for antigen retrieval, were neutralized with PBS. Sections were treated with blocking buffer (cat. no. 0634964, Blocking One Histo, Nacalai Tesque) and incubated with anti-Arl8A (1:50; HPA038759, Atlas) or anti-Arl8B (1:50; 13049-1-AP, Proteintech, Rosemont, IL) antibodies along with anti-α-Synuclein (1:250; pSyn#64, FUJIFILM Wako) and anti-Tyrosine hydroxylase (1:1000; ab76442, Abcam) overnight at 4°C. Primary antibodies were visualized with secondary antibodies (1: 1,000, Alexa Fluor405, 488, 594, and 647, Thermo Fisher Scientific) for 1 h at RT. To reduce lipofuscin autofluorescence, sections were further treated with TrueBlack Lipofuscin Autofluorescence Quencher (cat. no. 23007, Biotium, Inc. Fremont, CA), diluted with 70% ethanol for 30 s, and briefly washed with PBS before mounting with VECTASHIELD mounting medium (H1800, Vector laboratories, Newark, CA) and assessed using LSM880 with the Airyscan laser-scanning microscope system (Zeiss).