Scion image 4

The transgenic AD model and WT control mice were anesthetized and rapidly perfused and fixed with 4% paraformaldehyde. The brains were removed and placed in 4% paraformaldehyde for 24 h, and after dehydration in 30% sucrose, sectioned into slices 20 μm thick. After antigen repair and blocking, the slices were incubated with an anti-myeloperoxidase (MPO) antibody (1:1,000) at 4°C and gently oscillated overnight. Following second antibody incubation and wash, the nucleus was stained with hematoxylin. Then after DAB color rendering and sealing, the slices were observed under a microscope (Olympus BX43, Japan), and the images were captured and stored. The integral optical density (IOD) of the MPO stains of neutrophils per area was quantified with the Scion Image 4.0 software (Scion Inc., Fredrick, MD).

After treatment, the mouse was anesthetized with chloral hydrate and continuously perfused through the left ventricle with 4% polyformaldehyde for 0.5–1 h. Then the whole brain was isolated and immersed into 4% polyformaldehyde for 24 h. The brain was sliced using oscillating slicer (Leica) and the brain slices were rinsed with 10 mM PBS. Then the slices were treated by permeabilization with 10 mM PBS containing 0.3% Triton X-100 (v/v) for 30 min, and then blocked with 3% BSA-PBS (w/v) for 1 h, and incubated with 1:50 anti-c-Fos and 1:50 anti-NeuN antibodies in 10 mM PBS containing 0.3% Triton X-100 and 1% BSA and 2% goat serum (v/v) overnight at 4°C. After washes with PBS, slices were incubated with 1:100 Rhodamine-conjugated goat anti-rabbit and FITC-conjugated goat anti-rat secondary antibodies in 10 mM PBS containing 0.3% Triton X-100 and 1% BSA and 2% goat serum for 1 h at room temperature. Finally, the slices were mounted on glass slides with 30% glycerin, and visualized and captured with a fluorescent microscopy (Olympus BX43, Japan) for the expression and co-localization of c-Fos and neuronal marker NeuN. The fluorescence quantification software is Scion Image 4.0 (Scion Inc., Fredrick, MD, United States). We took the c-Fos/NeuN ratio of their fluorescence intensity of each single cell as data to make comparison between the vehicle and GYY-treated groups.

Protein concentration was determined using Bradford Protein Assay Kit (BioRad, Hercules, CA, United States). Fifteen micrograms of protein samples were run on a resolving 10% acrylamide/polyacrylamide gel and transferred to a PVDF membrane. All primary antibodies (anti-PSMA diluted 1:1,000; anti-PRMT2 diluted 1:500; anti-PARP diluted 1:1,000; anti-PARG diluted 1:1,000; anti-CPT1alpha diluted 1:1,000; anti-PGC1alpha diluted 1:1,000; anti-βactin diluted 1:5,000) from Cell Signaling Technology (Danvers, MA, United States) were incubated at 4°C overnight. After washing, membranes were incubated with the secondary antibodies (1: 5,000 diluted horseradish peroxidase (HRP)-anti-mouse (Sigma-Aldrich (St. Louis, MO, United States) and 1:1,000 diluted anti-rabbit IgG (Cell Signaling Technology (Danvers, MA, United States). The bands were visualized by a chemiluminescence reagent (Cell Signaling Technology). Gel imaging Chemidoc MP System (BioRad, Hercules, CA, United States) was used to visualize and examine the protein bands. Bands were quantified using Scion Image 4.0 (Scion Corporation, Chicago, Illinois, United States).

Intestinal epithelial cells were lysed in buffer containing 0.1 M Tris-HCl (pH 7.5)/2% SDS/10% glycerol/5% 2-mercaptoethanol, boiled at 95 °C for 5 min, centrifuged at 15,000 rpm for 5 min, and analyzed through reducing SDS-PAGE. Immunoblotting was performed using antibodies against occludin (Abcam, Cambridge, UK, ab15098, 1:100), myeloperoxidase (MPO, Abcam), and β-actin (Santa Cruz, TX, USA, sc-1615, 1:200). Quantitative analysis of Western blotting was performed using the Scion Image 4.0 (Scion Corporation, Frederick, MD, USA) and relative intensities of the target proteins to β-actin were shown.

To investigate the effect of CCCP on energy production in mitochondria, phosphocreatine (PCr) and ATP levels were measured using in situ³¹P magnetic resonance (MR) spectroscopy at 30 min after i.p. injection of 4 mg/kg CCCP or vehicle (CCCP: n = 5, vehicle: n = 4). Experiments were performed as previously reported [21 (link)]. Briefly, the animals were anesthetized with an i.p. injection of pentobarbital (30 mg/kg). Gradient echo transverse hydrogen-1 (¹H) MR images were obtained to define regions of interest. ³¹P MR spectroscopy of the anterior left ventricular wall was conducted using a surface coil (20-mm diameter) and depth-resolved surface coil spectroscopy [28 (link)]. Phosphorus metabolism was calculated by computer integration of the areas under the respective peaks (Scion Image 4.0, Scion Corporation, Frederick, MD, USA).

Western blotting was performed as described previously [41 (link)]. SDS-PAGE electrophoresis, transfer to nitrocellulose membrane and immunoblotting with ECL (Thermo Scientific) detection were performed according to standard manufacturer’s protocols (Bio-Rad Laboratories). Antibodies against the following proteins were used: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (clone 14C10), phospho-p53 (Ser15), p21 (clone 12D1), phospho-Rb (Ser807/811), HMGB1 (clone D3E5), as well as horseradish peroxidase-conjugated goat antirabbit IgG. Dilution rates were 1:1000 for all primary antibodies and 1:7000 for secondary ones. All antibodies were purchased from Cell Signaling. The Scion Image 4.0 (Scion Corporation) was used to select and determine the background-subtracted density of the bands in all the gels and blots.