Gfap antibody

GnRH antibody (ab16216) and IL-1β antibody (ab9722) were purchased from Abcam (Cambridge, UK). NF-κB p65(D14E12, Catalog number:8242) was purchased from Cell Signaling Technology, Inc (Boston, USA). Iba1 antibody (Catalog number: 10904-1-AP), GFAP antibody (Catalog number:16825-1-AP), SIRT1 antibody (Catalog number:13161-1-AP), TNF-α antibody (Catalog number:17590-1-AP), GAPDH (Catalog number:60004-1-Ig) were purchased from Proteintech Group Inc (Wuhan, China). Total cholesterol assay kit (Catalog number:A111-1-1), Triglyceride assay kit (Catalog number:A110-1-1), Low-density lipoprotein cholesterol assay kit (Catalog number:A113-1-1), High-density lipoprotein cholesterol assay kit (Catalog number:A112-1-1) and Glucose assay kit (Catalog number:F006-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA Kits of mouse MMP-2, MMP-9, TNF-α and IL-1β (The catalog numbers: EK0460, EK0466, EK0527, EK0394) were purchased from BOSTER Biological Technology.co.ltd (Wuhan, China). High-fat diet (MD12015) was purchased from Jiangsu Medison Biomedical Co., Ltd (Yangzhou, China). Elisa kit of SIRT1 was purchased from Enzyme-free Biotechnology Co., Ltd (Jiangsu, China).

IF was conducted as previously described,³⁶ (link) with some modifications. The frozen sections of the samples from different groups were incubated in normal goat serum and then with a mixture containing a TNIK antibody (sc-377215, mouse polyclonal antibody, 1:100), a glial fibrillary acidic protein (GFAP) antibody (rabbit polyclonal antibody, 1:100, Proteintech), and a microtubule-associated protein 2 (MAP2) antibody (chicken polyclonal antibody, 1:1000, GeneTex) overnight at 4 °C. Sections were washed and incubated with a mixture of DyLight 488-conjugated goat anti-mouse IgG (1:1000, Abcam), DyLight 405-conjugated goat anti-rabbit IgG (1:1000, Abcam), and DyLight 555-conjugated goat anti-chicken IgG (1:1000, Abcam) in a darkroom for 120 min at 37 °C. After washing, the sections were mounted with 80% glycerol. Fluorescence was detected using laser scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Germany) on an Olympus IX 70 inverted microscope (Olympus, Japan) equipped with a Fluoview FVX confocal scan head.

One day after behavioral testing, six rats per group were anesthetized with sodium pentobarbital (150 mg/kg, i.p.) and quickly decapitated for western blot analysis. Briefly, DG tissue was homogenized in ice-cold RIPA lysis buffer with a cocktail of protease/phosphatase inhibitors. The homogenate was centrifuged at 14,000 × g for 10 min at 4°C, and supernatants were collected. Protein concentrations in DG hippocampus was determined using BCA Protein Assay kits (Beyotime, China). Equal amounts of protein (30 μg) was loaded onto 8–15% SDS-PAGE gels for electrophoretic separation, then transferred onto PVDF membranes. Membranes were blocked in 5% non-fat milk for 1 h and probed with the following primary antibodies: Iba-1 antibody (1:1,000; WAKO, Japan), GFAP antibody (1:1,000; Proteintech, United States), anti-caspase-9 (1:200; Abcam Co., United Kingdom), anti-cleaved caspase-3 (1:500; Abcam Co., United Kingdom), or anti-β-actin (1:8,000; Santa Cruz Biotechnology). Secondary antibodies were horseradish peroxidase conjugated to mouse anti-rabbit/mouse immunoglobulin G (1:10,000; Sigma-Aldrich). Protein band densities were quantified using ImageJ software (NIH, Frederick, MD, United States) and were normalized to β-actin. Final data were expressed as a percent of that obtained from the control group.