Ab33922

The expression of the astrocyte marker GFAP on U87 cells (at a density of 5.0×10⁴ viable cells/cm²) cultured with 0, 1, 10 and 20 µM Aβ_1–42 for 48 h at 37°C was detected using immunofluorescence. Cells were washed with 1X PBS three times, fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and treated with 1X PBS containing 0.5% Triton-X-100 for 15 min on ice. Subsequently, blocking was performed using 10% normal goat serum (ab7481; Abcam, Cambridge, UK) for 1 h at room temperature. Following blocking, cells were incubated with rabbit monoclonal anti-GFAP (1:600; ab33922; Abcam) at 4°C overnight and were washed three times with 1X TPBS for 10 min. Goat anti-rabbit IgG H&L (Alexa Fluor 488; 1:400; ab150077; Abcam) secondary antibody was added for 1 h in a dark room at room temperature, followed by washing three times with 1X TPBS for 10 min. Photos were taken using a fluorescence microscope at ×100 magnification (Eclipse Ti-S; Nikon Corporation, Tokyo, Japan).

Total protein was extracted in cell lysate on ice and the concentration of proteins was tested using the bicinchoninic acid assay kit (Thermo Scientific Pierce, Rockford, IL, USA). Next, the proteins were separated by SDS‐PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were washed with Tris‐buffered saline‐tween (TBST) three times (15 min/time) and blocked with 5% skim milk for 2 h. Afterward, the membranes were cultured with the primary antibodies at 4℃ overnight: brain‐derived neurotrophic factor (BDNF) (ab108319, 1:1000, Abcam Inc., Cambridge, MA, USA), cAMP‐response element‐binding protein1 (CREB1) (ab178322, 1:500, Abcam), GFAP (ab33922, 1:500, Abcam) and MAP2 (ab11267, 1:1000, Abcam). Following washing with TBST (3 times/15 min), the membranes were cultured with the secondary antibody IgG (1:2000, ab205718, Abcam) for 2 h and then washed with TBST (three times/15 min) before chemiluminescence developing and visualization. The image of protein blotting was analyzed by Image J2x v2.1.4.7 software (Rawak Software, Inc. Dresden, Germany).

Mice were perfused with normal saline followed by a fixative reagent containing 4% paraformaldehyde in 0.1 M PBS; brain samples were harvested and protected in sucrose solution (30%) overnight and embedded in optimal cutting temperature compound (Solarbio, China) at −80°C. Then, 4-μm-thick coronal sections from of the brains were made in a cryostat, we sliced the entire brain but just focused on the hippocampus in this experiment. Sections were blocked in 5% BSA for 1.5 hours at room temperature and incubated in primary antibodies at 4 °C overnight. For double immunofluorescent staining, rabbit anti-NeuN (1:200; ab177487; Abcam) and mouse anti-NeuN (1:200; Millipore; MAB377) for neurons, rabbit anti-IBA1 (1:50; 10904-1-AP; Proteintech) and mouse anti-IBA1 (1:200; GT10312; GeneTex) for microglia, mouse anti-GFAP (1:200; SC-33637; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:200; ab33922; Abcam) for astrocytes were used as specific cellular markers. The sections were then incubated with Alexa Fluor 488 (1:200) and Alexa Fluor 594 (1:200; Invitrogen) for 1.5 hours at 37 °C. The staining was examined with a fluorescence microscope (OLYMPUS, BX53 MicroPublisherTM 5.0 RTV, Japan).

The neurons and astrocytes were washed with PBS three times and fixed with 4% paraformaldehyde for 10 min. Then, the cells were washed with PBS three times and permeabilized with Triton for 10 min. Following PBS washing three times, the cells were blocked with 0.5% bovine serum albumin for 30 min and cultured with the primary antibodies GFAP (ab33922, 1:2000, Abcam) and MAP2 (ab221693, 1:1000, Abcam) at 4℃ overnight. On the next day, the cells were cultured at room temperature for 15 min, washed with PBS three times, and cultured with the secondary antibody fluorescein isothiocyanate (ab6785, 1:10000, Abcam) in the dark at 37℃ for 90 min. Following PBS washing three times, the cells were sealed with fluorescent mounting media and observed under the fluorescence microscope (Olympus, Tokyo, Japan).