Supplementary Methods. Li J.-Y. et al. Lewy bodies in grafted neurons in Parkinson s patients suggest host to. graft disease propagation

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5 1 Supplementary Methods Li J.-Y. et al. Lewy bodies in grafted neurons in Parkinson s patients suggest host to graft disease propagation Neural transplantation and clinical assessment Detailed information was published previously and can be found in these papers 1-5 Immunosuppressive regimen Triple drug immunosuppression was given with corticosteroids, azathioprine and cyclosporine A (maintenance dosages were 0.1 mg/kg corticosteroids, 1 mg/kg azathioprine and 2 mg/kg cyclosporine A) prior to the first transplantation and months after the last surgery (in total 65 and 40 months of treatment in patient 3 and 8, respectively). No complications were noted in any of these patients. Autopsy and postmortem brain preparation Patient 3. The patient died of acute aspiration and subsequent cardiac arrest due to advanced PD. A post-mortem analysis, including neuropathologic examination of the entire brain was conducted. Tissue was prepared for specific analyses within the frames of the post transplantation follow-up study following procedures approved by the Regional Ethical Review Board in Lund. The brain was removed and fixed in 6% buffered formaldehyde solution for 2 months. Most of the basal ganglia and the left mesencephalon were sliced into mm thick blocks for frozen section preparation. The remaining brain slices, including small sections from the basal ganglia, right mesencephalon and other specific locations, were paraffin embedded for subsequent sectioning. Five µm thick sections were stained with hematoxylin-eosin for routine morphology and with Luxol fast blue with Nissl counterstaining for myelin and intracellular structures.

6 2 Patient 8. The patient died of aspiration pneumonia. After death, the brain was donated to the Queen Square Brain Bank for Neurological Disorders following procedures approved by a Multi-Centre Research Ethics Committee. The brain was bisected in the sagittal plane and the left half brain flash frozen. The right half brain was fixed in 10% buffered formalin, sliced in the coronal plane and tissue blocks selected for histology were processed into paraffin wax. Immunohistochemistry Brain blocks from the basal ganglia and ventral mesencephalon of patient 3 were serially cut in 40 µm thick sections on a cryostat. For diaminobenzidine - (DAB; Vector Lab. Inc., Burlingame, CA) staining, free floating sections were quenched in 3% H 2 O 2 and 10% methanol for 15 minutes before blocking (5% normal horse or goat serum, 0.3% triton-x-100 in 0.1M PBS, ph 7.4) for 1 hour. Sections were incubated in primary antibody (in 2% serum, 0.3% Triton-X-100) overnight at room temperature. Antibodies used are listed in Table 1. After rinsing, incubation in secondary antibody was performed (Biotinylated Horse-anti-Mouse or Goat-anti-rabbit, 1:200, Jackson Lab, West Grove, PA), ABC-solution (Vector Lab) and finally DAB for 40 seconds. Immunoreactive signals were assessed using a ScanScope CS (Aperio, Vista, CA). For double-labeling, two antibodies made in different animal species (rabbit, mouse or sheep) were combined and fluorescent Cy-2 or Cy-3 labeled secondary antibodies (Jackson) were used. To block autofluorescence, the sections were incubated in 5 mm CuSO 4 in 50 mm ammonium acetate (ph 5.0) for 90 min prior to mounting.

7 3 Tissue sections (20 m) from patient 8 were cut and stained using haematoxylin and eosin and immunohistochemical staining was performed using a standard avidin-biotin technique (for primary antibodies see Table 1). Antigen retrieval procedures Frozen or paraffin sections were treated with 10 mm sodium citrate solution to achieve effective antigen retrieval. Briefly, frozen free-floating sections were incubated in citrate solution (ph 8.5) at 80 C in a water bath for 30 minutes. Paraffin sections were mounted on capillary glass slides and treated in a microwave oven in citrate buffer at ph 6.0 for 15 minutes at 800 W. The sections were washed three times with 0.1 M PB and then followed by the protocol used for immunohistochemistry (see above). In patient 8, immunohistochemical staining for -synuclein and phosphorylated -synuclein required pretreatment of the tissue sections in concentrated formic acid and pressure cooking in citrate buffer at ph 6.0. Cell counting Numbers of surviving dopaminergic neurons were assessed around each injection track in frozen sections. The formula of Abercrombie was used to estimate the total number of TH-positive neurons per injection track 6. The average cell soma diameter (the mean length of the long- and short-axis) of the transplanted dopaminergic neurons was estimated from 30 randomly selected neurons on each side, which received transplants at different times.

8 4 Table 1. Antibodies used in this study Antibody name Species Source Working dilution TH Rabbit Pel-freeze 1:500 TH (MAB 318) Mouse Chemicon 1:2000 TH (AB1542) Sheep Chemicon 1:500 -synuclein Rabbit Chemicon 1:200 (AB5038P) -synuclein (Clone Mouse Zymed 1:600 LB509) CD68 (Clone PG- Mouse Dako 1:200 M1) -synuclein Mouse Vector 1:75 Ser-129 Rabbit Professor Takeshi 1:100 phosphorylated - synuclein Iwatsubo tau (AT8) Mouse Autogen Bioclear 1:600 neurofilaments Mouse Novocastra 1:50 (clone RT 97) p62 Mouse BD Transduction 1:200 ubiquitin Rabbit DAKO 1:200 ubiquitin (AB1586) Sheep Chemicon 1:2000 Girk2 Rabbit Alomone Labs 1:100 Calbindin D28k Mouse Sigma-Aldrich 1:1000 Iba1 Rabbit Wako Chemicals 1:500 References 1. Lindvall, O., et al. Prog Brain Res 82, (1990). 2. Lindvall, O., et al. Ann Neurol 35, (1994). 3. Hagell, P., et al. Brain 122, (1999). 4. Piccini, P., et al. Nat Neurosci 2, (1999). 5. Piccini, P., et al. Ann Neurol 48, (2000). 6. Abercrombie, M. Anat Rec 94, (1946).