Pva dabco

Cells were transduced with the LV-XEP-2Gi2R carrying the IRES-linked sequences for 2xGFP:NES and 2xRFP:NLS. We used the HIV-Rev NES (LQLPPLERLTL) and the SV40 Large-T NLS (PKKKRKV). For imaging, cells were transferred to tissue culture-treated ibidi μ-slides coated with astrocytes for iNs, PluriPro matrix (Cell Guidance Systems) for iPSCs, and uncoated for fibroblasts. Cell were fixed with 4% PFA for 20 min at room temperature and mounted in PVA-DABCO (Sigma Aldrich). Confocal images were taken on Zeiss LSM710 or LSM780 series confocal microscope. All data for one NCC experiment were acquired from cells cultured, transduced, and processed in parallel and images were taken on the same microscope with exactly the same settings reused. For analysis, 2 μm confocal sections through the nuclear layer were acquired from three confocal z stacks. Image J software was used to identify nuclear ROIs based on binarized DAPI channels and green and red fluorescence means were measured in the ROIs. Because of the morphological complexity of the astrocyte iN co-cultures, neuronal nuclear ROIs were selected manually. To quantify the integrity of cellular NCC, GFP_nuc/RFP_nuc were calculated in transgenic cells using Microsoft Excel and further processes in GraphPad Prism 6.

C6 cells were transfected with the empty vector or a construct of overexpression for mAtg5 wild type, or mutated nonfunctional mAtg5. Cells were exposed during 20 h to 0.5 uM Aβ1–42 peptide (Sigma) previously incubated for 72 h with distilled water at 37°C to allow fibrils formation.
After exposure, cultures were washed twice with PBS, fixed with 4% paraformaldehyde/4% sucrose in PBS 30 min at RT and washed five times with PBS. Fixed samples were permeabilized with Triton X-100 0.25% in PBS (10 min at RT), washed three times with PBS and blocked with 1% BSA in PBST (Tween 20 0.1% in PBS) overnight at 4°C. Coverslips were incubated with a solution of goat anti-LC3 (Santa Cruz Lab) 1:100 and mouse anti-Aβ (clone 4G8, Covance) 1:1000 for 1 h, washed three times with PBS, and incubated with a solution of Alexa 488 donkey anti-goat (Life Technologies) 1:1000 in 1% BSA in PBST; 1 h. After three washes with PBS, samples were incubated with Alexa 555 goat anti-rabbit (Life Technologies) 1:1000, 1 h. Finally, coverslips were washed three times with PBS and mounted with PVA-DABCO (Sigma-Aldrich, USA). Representative images were obtained by Nikon Eclipse E80 confocal scanning laser microscope (Nikon Instech Co., Ltd., Karagawa, Japan). Negative controls were performed by incubating samples under the same conditions without one or both primary antibodies (data not shown).