Ab1783

Hippocampi were lysed in RIPA buffer with 1× complete protease inhibitors (cOmplete, EDTA‐free Protease Inhibitor Cocktail, Roche). Protein levels were assessed with a Bradford assay with bovine serum albumin as the standard.
About 20 µg of denatured proteins in Laemmli buffer were separated by 8% SDS‐polyacrylamide gel electrophoresis and blotted onto PVDF membranes (GE Healthcare). Nonspecific binding was blocked with PBS‐0.1% Tween‐20 (PBST) with 3% (w/v) non‐fat dried milk (Sigma), for 1 hr at 20–25°C. Membranes were incubated overnight at 4°C in PBST with 3% milk and primary antibodies: anti‐GLT1 (1/500, Millipore AB1783), mouse anti‐β‐Actin (1:5000, Sigma A1978). Membranes were then incubated at 20–25°C for 1 hr in PBST/3% milk with POD‐conjugated recombinant protein‐A (Invitrogen). Protein bands were detected by enhanced chemiluminescence (GE Healthcare) and quantified by densitometry with ImageJ. Protein levels were normalized to those of β‐actin controls.

Five micron sections were cut and placed onto slides. Immunofluorescence was performed by first removing the paraffin and then rehydrating the sections. After that, antigen retrieval was performed (Antigen unmasking solution, Tris-based, Vector Labs cat#H-3301). Sections were permeablized with 0.4% Tritonx-100-PBS, blocked with 10% normal goat serum (NGS) in PBS with 0.4% Triton X-100, and then incubated with primary antibody in blocking solution NeuN (1:200, Thermo Fisher, 26975-1-AP) ARG1 (1:200, Santa Cruz, SC-18354), GFAP (1:200, Invitrogen, MA5-12023), IBA1 (1:200, Invitrogen, MA5-41621) GLT-1 (1:200, AB1783, Sigma-Aldrich, NRF2 (1:200, Invitrogen, PA5-27882) followed by secondary (1:400 sary), and then DAPI (1:400 Thermo Fisher Scientific Cat#62248). Fluorescence was counted from three images per high-powered field (HPF) at 20× and quantified using ImageJ software. TUNEL stain was performed according to the manufacturer’s instructions (Sigma, ApopTag Fluoresce in in situ Apoptosis Detection Kit, S7100) and was detected with Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor^® 488 Conjugate, Cell Signaling cat #9669). n = 10 control and n = 10 drug treated brains at each time point.

Cultured hippocampal cells were incubated with hMSC-EVs (6 × 10⁸ particles) for 22 h after addition of vehicle (PBS containing 2% DMSO) or AβOs (500 nM) for 2 h. In uptake experiments, we employed hMSC-EVs derived from hMSCs that were double-labeled with SYTO RNASelect (for RNA labeling) and Vybrant DiI (for membrane labeling) (both from Molecular Probes). Cells were fixed with 4% paraformaldehyde/4% sucrose solution for 15 min at 4 °C. Cell nuclei were labeled with DAPI. Images were acquired on a Zeiss LSM 510 confocal microscope.
For immunocytochemistry, fixed cells were blocked with 4% bovine serum albumin at room temperature. Primary antibodies used were rabbit anti-MAP 2 (1:200; Santa Cruz), mouse anti-GLUA1 (1:200; MAB2263, Santa Cruz), and guinea pig anti-GLT-1 (1:400; AB1783, Millipore) and were incubated overnight at 4 °C. For labeling with mouse anti-synaptophysin (1:1000; Vector Labs) and rabbit anti-PSD-95 (1:200; Santa Cruz) antibodies, cells were permeabilized with 0.1% Triton X-100 (Merck) for 5 min at room temperature before blocking with 10% horse serum for 1 h. After incubation with primary antibodies, cells were washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:1000; Thermo Fisher) for 2 h at room temperature. Images were acquired on a Zeiss Axio Observer Z1 microscope or a Nikon C2 confocal microscope.