Reagents and antibodies
Protein isolation with RIPA lysis buffer was purchased from Elpis biotech (#EBA-1149, Deajeon, Korea) and Pierce™ BCA Protein Assay Kit was purchased from Thermo (#23227, MA, USA). Anti-GFAP, rabbit polyclonal antibody was purchased from DAKO (#Z0334, CA, USA). Anti-NeuN rabbit monoclonal antibody was purchased from Millipore (#3838, MA, USA). Anti-PSD-95, mouse monoclonal antibody, anti-rabbit, goat polyclonal tagged Alexa Fluor 488, anti-mouse, goat polyclonal tagged Alexa Fluor 555 and 4’. 6-diamidino-2-phenylinodole (DAPI) were purchased from ThermoFisher (#MA1-046, #A11034, #A11012, #A21426 and #D3571, MA, USA). Anti-GAPDH, rabbit polyclonal antibody was purchased from AbFrontier (#LF-PA0018, Seoul, Korea). Rabbit polyclonal antibody was purchased from WAKO (#016-20001, Osaka, Japan). Anti-mouse, sheep polyclonal horseradish peroxidase (HRP) tagged antibody was purchased from Abcam (#ab26116, #ab26113 and #ab6808, EA, UK). Anti-p-STAT3 (Tyr 705), STAT3, p-p44/42, p44/42 rabbit polyclonal antibody was purchased from Cell signaling (#9145, #12640, #4377 and #4695 MA, USA). Anti-NF-κB mouse monoclonal were purchased from Santa Cruz (#sc-7151, Taxas, USA)
Experimental animals
All of the experimental procedures were approved by the Animal Care Committee of Seoul National University (Approval number: SNUIBC-171011-2). Transgenic mice with 5XFAD mutations were purchased from Jackson Laboratories (strain: B6SJL-Tg [APP Sw, Fl, Lon, PS1, M146L, L286V] 6799Vas/J) and maintained by crossing hemizygous transgenic mice with B6SJL F1 mice. Six- month-old male mice were used for the experiments. The mice were housed in five per cage with a 12-hour light/dark cycle and ad libitum access to food and water under standard laboratory housing conditions.
Primary astrocyte culture
The primary astrocyte culture was described previously [15]. Briefly, primary mixed glial cultures were prepared from postnatal day 1 (P1) C57B/L6 mice. The cortex was dissociated with 0.25% trypsin and filtered with a cell strainer. The mixed glia was grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% (vol/vol) heat- inactivated FBS and penicillin (10 units/ml) and streptomycin (10 mg/ml) in a humidified cell incubator (Binder, Germany) at 37°C under a 5% CO2 atmosphere. When the mixed glia filled 80% of the T75 plate, astrocyte and microglia were isolated via a 250 rpm shaking incubator at 37°C for overnight. Microglia were detached from the plate after shaking isolation. The astrocytes were detached with 0.25% trypsin-EDTA and seeded onto poly-L-lysine coated plates for the experiment.
Contextual fear conditioning
Contextual fear conditioning was tested as previously described [8]. Briefly, prior to training, WT and 5XFAD mice were placed into the chamber for 10 min for habituation. Each mouse was placed in the conditioning chamber (13 × 13 × 25 cm) for 3 min and acquired three repetitions of a foot-shock (0.7 mA, 2 sec) at 1 min intertrial intervals during the training day. On the next day, trained mice were placed in the same chamber, and the freezing behavior was measured over periods of 3 min. The freezing behavior was defined as immobility except for respiratory movements. The total freezing time was analyzed as a percentage in the test period. The control group was habituated to the chamber without shock. The 1 hour group received electric shock and was sacrificed after 1 hour. The 24 hour group received electric shock and sacrificed the next day.
T-maze test
T-maze test was performed as previously described [16]. Briefly, prior to training, WT and 5XFAD mice were placed into deem light to habituation for 1 hour. The testing mice was positioned at the starting zone. After the mice choose one way, the way was blocked and the mice stay for 30 sec. During clean the T-maze with 30% EtOH, the mice stayed in the home cage for 1 min and return into the starting zone. If the mice chose the other way, it will get 1 point. If the mice chose the same way, it will get 0 point. This trial performed after 30 min and the next day.
Western blotting
Western blotting analysis was described previously [16]. Briefly, the mice were anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) and the dissected brain tissues were stored at -80°C before protein lysis. Hippocampi were homogenized with RIPA mix. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 5,000), anti-NeuN (rabbit, 1: 1,000), anti-p-STAT3 (rabbit, 1:1,000), anti-STAT3 (rabbit, 1:1,000), anti-p-p44/42 (rabbit, 1:1,000), anti-p44/42 (rabbit, 1:1,000), anti-p-p65 (rabbit, 1:1,000), anti-p65 (rabbit, 1:1,000), anti-PSD95 (mouse, 1:2,000), anti-GAPDH (rabbit, 1:10,000). Secondary antibodies were conjugated with HRP (1: 2,000). The HRP signals were visualized using an enhanced chemiluminescent substrate.
Immunofluorescence
The protocol was previously described in the paper by Choi et al [8]. The brains were perfused with heparin dissolved in PBS (pH 7.2) for 5 min were fixed with 4 % paraformaldehyde for 24 hours. The fixed brains were transferred into 30 % sucrose solution for 48 hours. The brain tissue was sectioned into 30 μm thick slices with a cryotome with the chamber at -20°C and the bar temperature at -25°C.
For GFAP and NeuN staining, the brain sections were heated in a 95°C water bath for 30 min with 10mM citrate acid (pH 6.0) for antigen retrieval. The sections were blocked with 2 % BSA and 0.3 % Triton X-100 in PBS for 1 hour. Primary antibodies GFAP (1:1,000) and NeuN (1:500) were applied overnight at 4°C. Secondary antibodies, anti-rabbit-488 (1:200) and anti-mouse-555 (1:200) were applied for 2 hours at room temperature. The stained brain tissue samples were imaged with confocal microscopy using LSM 510 (Carl Zeiss, Germany).
Statistical analysis
Data are expressed as the mean ± SEM (means ± standard error of the mean). One-way ANOVA followed by Tuckey post hoc analysis or Student’s t-test (SPSS, IL, USA) was performed to determine statistical significance. The results were considered to be statistically significant at p<0.05.