Anti goat alexa 647

At the time of autopsy, different organs for each mouse were sampled and either fixed in zinc–formalin or 4% paraformaldehyde and processed as described in Appendix Supplementary Materials and Methods. Immunohistochemical staining was performed utilizing the following antibodies: anti‐F4/80 (clone A3‐1, AbD Serotec); anti‐RFP (rabbit polyclonal, ab62341 AbCam); anti‐CD34 (clone MEC14.7, Biolegend); anti‐Ki67 (clone SP6, Neomarkers); anti‐CD3 (clone SP7, AbCam); and anti‐CD45R/B220 (clone RA3‐6B2, BD Pharmingen). All images were acquired using the Aperio Scanscope CS2 system (Leica Biosystems). Immunofluorescence staining was performed utilizing the following antibodies: anti‐GFP (rabbit polyclonal, A11122 Invitrogen) + anti‐rabbit Alexa 488 (Invitrogen); anti‐MMR (goat polyclonal, AF2535 R&D Systems) + anti‐goat Alexa 647 (Invitrogen); anti‐F4/80‐PE (clone A3‐1, AbD Serotec); anti‐CD11b‐Alexa 647 (clone M1/70; Biolegend); and Hoechst 33342 (Invitrogen). Confocal images were acquired using a Leica TCS SP2 or SP8 confocal system (Leica Microsystems) that are available at the SRSI Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC).

To identify direct- and indirect-pathway SPNs electrodes were loaded with biocytin, as described (Martella et al., 2009 (link)). Briefly, slices were fixed with 4% PFA in 0.12 M PB and 30 μm thick sections were cut from each slice with a freezing microtome, then dehydrated with serial alcohol dilutions to improve antigen retrieval and reduce background (Buchwalow et al., 2011 (link)). We used the following primary antibodies: goat anti-DARPP-32 (1:500 AF6259, R and D system), mouse anti-Enkephalin (1:1000 MAB350, Millipore), and secondary antibodies: anti-goat alexa 647 (Invitrogen), anti-mouse cyanine 3 (Jackson ImmunoResearch) and streptavidin-conjugated alexa 488 (Life Technologies). All sections used for analysis were processed together. Images were acquired with a LSM700 Zeiss confocal laser scanning microscope and analyzed with ImageJ software (NIH; Schneider et al., 2012 (link)). Noise was reduced by applying background subtraction in ImageJ.

TSC were seeded in a 12-well plate and incubated at stem cell conditions for ~72 h. GM1a incorporation was performed in selected wells as described above. Afterwards, TSC were washed two times with serum-free TSC medium. Incubation with human complement FH (Complement Technology, Texas, USA) was carried out at 37 °C for 30 min in serum-free TSC medium at a final concentration of 10 µg/ml. Subsequently, TSC were directly fixed as described above and used for indirect immunofluorescence analysis. For FH binding to PNH-RBC, 50 µl of 5*10⁷ erythrocytes/ml were treated with our without 100 µM GM1a for 60 min at 37 °C and subsequently incubated with 100 µg/ml FH for 30 min at 37 °C. FH binding was assessed via flow cytometry using goat anti-FH and anti-goat Alexa 647 (Invitrogen).