2.10 Plasmid and Transfection
For overexpression of the IRAK1, BV2 cells were transfected with the plasmid pcDNA-3.1-IRAK1 (Hanheng Biotechnology, China) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. Plasmid pcDNA-3.1 was used as a control. In brief, BV2 cells were seeded in DMEM (10% FBS) and cultured until them until achieved 80% confluence. We then replaced the culture medium with low-serum media (0.5% FBS), and then BV2 cells were transfected with 10 µg/well of plasmid. Cells were collected for further study at 48 h post-transfection. The efficiency of IRAK1 overexpression was measured qRT-PCR as indicated in Fig. 8E.
2.11 Nitrite assay
Griess assay was employed to detect the accumulation of nitrite (NO2−) in cultured supernatant. Microglial cells were first planted into 96-well plates at the density of 5 × 104 cells/well, and then the cells were treated with LPS (100 ng/ml) for 24 h. Next, 50 µl Griess reagent and 50 µl culture supernatant fluids were mixed together for 15 min at 37 °C, and the absorbance was determined at 540 nm.
2.12 Luciferase reporter assay
Luciferase reporter assay was conducted as previously described. In brief, by employing Lipo-fectamine 3000 reagent (Invitrogen), BV2 cells were cotransfected with Renilla luciferase plasmid (pRL-SV40-C) and AP-1 luciferase reporter plasmid or NF-κB luciferase reporter plasmid for 48 h. Then, the BV2 cells were treated with LPS (100 ng/mL) for 24 h with or without galectin-1 (5 µg/ml). Reporter activity was detected using Dual Luciferase Reporter Gene Assay Kit (Beyotime, China) according to the manufacturer’s instructions.
2.13 MTT cell viability assay
MTT assay was used to measure the cell viability in strict accordance with the manufacturer’s instruction. In brief, cells administered with LPS for 6 h with or without galectin-1, were seeded at the density of 1 × 104 cells/well in 96-well plates and cultured for 24 h at 37 °C. MTT (Sigma-Aldrich, M2128) was added to the cells at a final concentration of 0.5 mg/mL, and the cells were incubated at 37℃ for another 4 h. Finally, cells were washed with PBS for three times and formazan crystals were dissolved in 100 mL DMSO. The absorbance was read at 490 nM using a microplate reader (PerkinElmer, USA) .
2.14 Western blot
Proteins in cell culture media and hippocampal tissues were lysed in RIPA lysis buffer with protease inhibitor cocktail. Protein samples were subjected to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were subsequently incubated with antibodies iNOS (Abcam #ab178945, USA, 1:500), IL-1β (Abcam #ab234437, 1:500), Il-6 (Abcam #ab233706, 1:500), TNF-α (Abcam #ab215188, USA, 1:500), c-Jun (CST #9165, USA, 1:500), p65(CST #8242, USA, 1:500), IRAK1 (CST #4504, USA, 1:500), β-actin (CST #3700, USA, 1:2000). Then the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and detected with enhanced chemiluminescence. The results were analyzed by using ImageJ software (National Institutes of Health, USA).
2.15 Immunofluorescence
The mice were sacrificed, and their brains were fixed in 4% paraformaldehyde at 4 °C for 24 h and then embedded in paraffin. The brain tissues were cut into 10-µm-thick slices. The slices were blocked with 10% donkey serum for 1 h, incubated with anti-Iba-1 (Abcam, #ab178847, 1:200) or anti-NeuN (CST #24307, USA, 1:200) antibodies overnight at 4 °C. After washing with PBS solution for three times, sections were incubated with secondary antibodies at room temperature for 1 h. After being washed, the sections were incubated with DAPI for nuclear staining. By using ImageJ software (National Institutes of Health, USA), Iba-1-positive cells and NeuN-positive cells were counted with a DAPI counterstain.
2.16 Statistical analysis
The sample size was calculated by using the SPSS 16.0 software (SPSS, Chicago, USA) to achieve an 80% power at a significance level of 0.05. Two‒way ANOVA was employed to evaluate latency in the MWM test and student's t test was used to assess the differences between two groups. Whether the data were normally distributed is detected by the Shapiro-Wilk normality test. The GraphPad Prism software (version 7.0, CA, USA) was employed to perform the statistical analyses. In all cases, statistical significance was accepted at P < 0.05.