Application suite las advanced fluorescence 2

FUW mCherry-GFP-LC3 was a gift from Anne Brunet (Addgene plasmid # 110060; http://n2t.net/addgene:110060; RRID:Addgene_110060). The concentrated lentivirus of mCherry-GFP-LC3 was added to reprogrammed MSNs at PID20. For imaging of cells expressing mCherry-GFP-LC3, cells were washed once with PBS, fixed and stained by TUBB3 antibody, after verification of expression of GFP and mCherry by microscopy at PID26. Images were captured using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS) Advanced Fluorescence 2.7.3.9723. For quantification of autophagosome (i.e., mCherry+, GFP+ puncta) and autolysosome (i.e., mCherry+, GFP− puncta) compartments in MSNs from multiple HD patients and control individuals, measurements were performed in cells having at least 3 puncta per cell.

Reprogrammed cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, #15710) for 20 min at room temperature (RT), and permeabilized with 0.2% Triton X-100 for 10 min at RT. Cells were blocked with 5% BSA and 1% goat serum in PBS and incubated with primary antibodies at 4°C overnight, then incubated with secondary antibodies for 1 hr at RT. Cells were incubated with DAPI (Sigma, D-9542) for 5 minutes followed by washing with PBS. Images were captured using a Leica SP5X white light laser confocal system with Leica Application Suite (LAS) Advanced Fluorescence 2.7.3.9723.