Darpp32

We measured the striatal density of DARPP‐32 (Millipore, 1:200)‐positive areas by immunostaining to investigate the neuroprotective effects of the transplanted cells. The DARPP‐32 immunostaining in the striatum was estimated using an optical fractionator and unbiased stereology of stained cells. To measure striatal density, three images of DARPP‐32 immunostaining near the middle of the striatum were captured. The images were delineated at 4× magnification, and then, the density of images was quantitated using Image J software (rsb.info.nih.gov, by W. Rasband).

Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 (Weiss et al., 2009 (link)) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4°C. Fluorescence conjugated secondary antibodies were used to reveal the binding of primary antibodies (Life Technology, cat. no. A21206, A31572, A21202, A21203, 1:1000) and nuclei were stained with DAPI (Sigma, cat. no. D9542, 1:1000). Images were captured by Leica TCS SP8 confocol system. Co-localization was quantified using the Pearson's correlation coefficient and the overlap coefficient, which were calculated with Image-pro Plus software. Data represent the mean with standard deviation. One-way ANOVA experiments were performed to judge the statistical significance.

For western blotting the following antibodies were used: total LRRK2 [MJFF2 (c41-2)] (Abcam, Cambridge, UK, Cat# ab133474, 1:300), phospho-S935 LRRK2 (Abcam, Cambridge, UK, Cat# ab133450, 1:500), phospho-S1292 LRRK2 (Abcam, Cambridge, UK, Cat# ab203181, 1:300), total Rab10 (Abcam, Cambridge, UK, Cat# ab104859, 1:500), phospho-T73 Rab10 (Abcam, Cambridge, UK, Cat# ab230261, 1:400), tyrosine hydroxylase (Millipore, Burlington, MA, USA Cat# AB152, 1:10000), DARPP32 (Millipore, Burlington, MA, USA Cat# AB10518, 1:10000) and β-actin (Sigma-Aldrich, St. Louis, MO, USA Cat# A2066, 1:10000). For immunofluorescence: β-tubulin-III (Sigma-Aldrich, St. Louis, MO, USA, Cat# T8578, 1:1000); total LRRK2 [MJFF2 (c41-2)] (Abcam, Cambridge, UK, Cat# ab133474, 1:200), phospho-S935 LRRK2 [UDD2 10(12)] (Abcam, Cambridge, UK, Cat# ab172382, 1:200), GLT1 (EMD Millipore, Burlington, MA, USA Cat# AB1783, 1:400), GFAP (Dako-Agilent, Santa Clara, CA, USA, Cat# Z0334, 1:400) CD11b [M1/70] (eBioscience™ from Thermo Fisher Scientific, Waltham, MA, USA, Cat# 14-0112-82, 1:200).