Mip 3α

Approximately 40 mg of brain tissue lysed in RIPA buffer including protease inhibitors were homogenized and centrifuged at 12,000 rpm for 5 min at 4 °C. The concentration of protein was detected by a BCA Protein Quantitative Kit (Dingguo Changsheng Biotechnology, Beijing, China). Equivalent amounts of protein samples (40 ng) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF). The antibodies used were as follows: APP 1:1000 (Abcam, ab126732, Cambridge, UK), SAA 1:1000 (Abcam, ab199030, Cambridge, UK), CYP27A1 1:1000 (Abcam, ab126785, Cambridge, UK), CYP7B1 1:1000 (ABclonal, A17872, Wuhan, China), CYP46A1 1:2000 (Abcam, ab244241, Cambridge, UK), RORγt 1:2000 (Abcam, ab207082, Cambridge, UK), Foxp3 1:1000 (Abcam, ab215206, Cambridge, UK), IL-17A 1:3000 (Abcam, ab189377, Cambridge, UK), GM-CSF 1:1000 (Proteintech, 17762-1-AP, Chicago, IL, USA), MIP-3α 1:1000 (Abcam, ab106151, Cambridge, UK), IL-10 1:1000 (Abcam, ab189392, Cambridge, UK), IFN-λ2 0.1 µg/mL (R and D, AF4635, Minneapolis, MN, USA). The protein density was measured by Image System Fusion FX (Vilber Lourmat, Paris, France) and GAPDH was used as the reference for standardization.

Tumor tissue of the nude mice was fixed in 10% formalin buffer for 1 day, and paraffin was used to embed the tissues; then, the fixed tissue samples were sectioned. After deparaffinization and gradient hydration, antigen retrieval was performed. Then, the sections were incubated with polyclonal anti-TLR4,COX-2,iNOS,IL-6,IL-8, MIP-3α, TGF-β1 and VEGF antibodies(1:500, Abcam, Cambridge, MA, UK) and placed in a 4°Crefrigerator overnight. The following morning, horseradish peroxidase (HRP)-conjugated anti-TLR4, COX-2,iNOS,IL-6,IL-8, MIP-3α, TGF-β1 and VEGF secondary antibodies (1:200; Arigobio, Taiwan, ROC) were used. Then, the sections were incubated in 3,3′-diaminobenzidine (DAB, Beyotime, Shanghai, China). Following counterstaining with hematoxylin, the sections were washed and dehydrated, and a cover slip was mounted with neutral balsam. Image-Pro Plus software (IPP; produced by Media Cybernetics Corporation, USA) 6.0 was used for the quantitative analysis of the IHC results.