Aβ 6e10

The cultured AD patient iNs were washed with  1× phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde and washed twice with 1× PBS containing 0.1% triton-X. Primary antibodies (anti-βIII-tubulin, 1:1000, Sigma-Aldrich, St. Louis, MO; NeuN, 1:500, Millipore, Darmstadt, Germany; MAP2, 1:200, Cell Signaling, Beverly, MA; VGLUT1, 1:200, Invitrogen, Grand Island, NY; Synapsin 1, 1:500, Invitrogen; Aβ (6E10), 1:500, Biolegend, San Diego, CA; Aβ42, 1:500, Biolegend; Phosphorylated tau, 1:400, Pierce, Rockford, IL;  apoE4, 1:500, Millipore; LC3B, 1:500, Cell Signaling; EEA1, 1:500, Millipore) were applied overnight at 4 °C. Appropriate secondary antibodies were obtained from Invitrogen and incubated for 2 h at room temperature. After washing, the samples were treated with 6-diamidino-2-phenylindole (DAPI, Invitrogen) and mounted in Fluoromount-G mounting medium. Representative images were taken on a Zeiss confocal microscope (Zeiss, Oberkochen, Germany, LSM800). An investigator blinded to the experimental conditions analyzed all tests. Image J software was used to analyze particles and to quantify immunofluorescent signals within regions of interest. These data were processed in parallel on the same confocal microscope with the same setting.

Confocal microscopy was used for semi-quantification and qualitative analysis. Brain samples were subjected to paraformaldehyde (4% in PBS) fixation, overnight, followed by cryoprotection with sucrose (30% in PBS). Cryosections, at 30 μm thick, were subjected to immunohistochemical analysis using antibodies against Aβ (6E10, 1:200, Biolegend), GFAP (astrocyte marker, 1:200, Abcam), p-STAT3 (1:200, Cell Signaling Technology), or sEH (1:100) for overnight incubation at 4 °C. The corresponding secondary antibodies conjugated with Alexa Fluor (Invitrogen) as indicated in the results were applied for 2 h. Tissues were coverslipped with mounting medium (Vector Lab) containing 4,6-diamiino-2-phenylindole (DAPI) for nuclei counterstaining. Images were acquired using a Leica confocal microscopy imaging system. GFAP immunoreactivity has been widely used for the evaluation of morphological alterations in activated astrocytes in vivo and thus the quantification of GFAP immunoreactivity was used as a measure of astrocyte activation in this study. Semi-quantification of the immunoreactivity from two sections per mouse was performed using MetaMorph imaging software.