Lysis buffer

Blood was collected from the hearts of mice at the determined end points and centrifuged at 3000 rpm to isolate the plasma by a microvolume high-speed cooling centrifuge MX-201 (TOMY, Tokyo, Japan) and stored at −20 °C. The dorsal skin, livers, and kidneys were excised and homogenized with lysis buffer (Kurabo, Osaka, Japan), and then, the tissue lysate was centrifuged at 10,000 rpm. Plasma concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, creatinine, hyaluronic acid, endostatine, glutamate oxaloacetate transaminase (GOT), glutamic acid pyruvate transaminase (GPT), matrix metalloproteinase (MMP)-1, histamine, and angiopoitin 1 and 2 were measured using appropriate ELISA kits according to manufacturers’ instructions (IL-6, Enzo Life Sciences, Farmingdale, NY, USA; TNF-α, hyaluronic acid, and angiopoietin 2, R&D Systems, Minneapolis, MN, USA; MMP-1, MyBioSource, San Diego, CA, USA; histamine, Bertin Pharm, Montigny-le-Bretonneux, France; creatinine, Cayman, Ann Arbor, MI, USA; Endostatine, BioMedica, Vienna, Austria; angiopoietin 1, EIAAB Science, Wuhan, China; GOT and GPT, Wako, Osaka, Japan; ROS, Cell Biolabs Inc., San Diego, CA, USA). Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Hippocampal tissue samples were homogenized in a lysis buffer (Kurabo, Osaka, Japan). The homogenates were centrifuged, and the supernatants were obtained. Western blotting was performed as previously described [24 (link)]. The membranes were incubated at room temperature for 1 h with primary antibodies against brain and muscle arnt-like 1 (Bmal1) (1:1000; Cell Signaling Technology, Danvers, MA, USA), Cryptochrome 1 (Cry1) (1:1000; Proteintech Group, Rosemont, IL, USA), Cry2 (1:1000; Proteintech Group, Rosemont, IL, USA), ionized calcium-binding adapter protein 1 (Iba1; marker of microglia (1:1000; Wako, Osaka, Japan), CC-Chemokine receptor 7 (CCR7) (1:1000; Abcam; Cambridge, MA, USA), and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) as loading controls. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were captured using Multi Gauge Software ver. 3.0 (Fujifilm, Greenwood, SC, USA).

Hippocampal tissue samples were collected on the final day of the experiment. The hippocampus was isolated and homogenized in a lysis buffer (Kurabo, Osaka, Japan). The tissue extracts were centrifuged (Tomy MX-201, TOMY DIGITAL BIOLOGY Co., Ltd., Nerima-ku, Tokyo, Japan) at 10,000 rpm, and supernatants were collected to perform the assay. Commercial enzyme-linked immunosorbent assay kits were used, according to the manufacturers’ instructions, to measure the following: iNOS and Arg-1 (CUSABIO, Houston, TX, USA), ANGPTL2 (biorbyt, Cambridge, UK), IL-6 (RayBiotech Life, Peachtree Corners, GA, USA), TNF-α (Enzo Life Sciences, Farmingdale, NY, USA), and IL-1β (Abcam, Cambridge, MA, USA). Optical density was measured using a microplate reader (Molecular Devices; Sunnyvale, CA, USA).

At the end of the study, the brains were harvested. Following this, the hippocampus was rapidly dissected, excised, and homogenized in lysis buffer (Kurabo, Osaka, Japan), and the lysate was centrifuged at 10,000 rpm. ELISA kits were used to measure the hippocampus concentrations of AGEs, tumor necrosis factor (TNF)-α, RAGE (MyBioSource, San Diego, CA, USA), cluster of differentiation (CD) 163 (Elabscience, Houston, TX, USA), ionized calcium binding adapter molecule 1 (Iba1: South San Francisco, CA, USA), CC-chemokine receptor 7 (CCR7: abbexa, Cambridge, UK), inducible nitric oxide synthase (iNOS), and arginase-1 (CUSABIO, Houston, TX, USA). NO was assayed using assay kits (NO₃⁻+NO₂⁻ colorimetric assay; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. The microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used for the measurement of optical density.

The dorsal skin samples were homogenized in lysis buffer (Kurabo, Osaka, Japan). Following this, the homogenates were centrifuged at 8000× g for 10 min, and the resultant supernatants were collected. Western blotting was performed as previously described [23 (link)]. Briefly, the membranes were incubated with primary antibodies against F4/80 (marker of macrophage; 1:1000; ThermoFisher Scientific, Waltham, MA, USA), chemokine receptor 7 (CCR7: marker of M1 type macrophage; 1:1000; Abcam, Cambridge, MA, USA), CD163 (marker of M2 type macrophage; 1:1000; ThermoFisher Scientific), or β-actin as a loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA). Immune complexes were detected using ImmunoStar Zeta reagent (Wako, Osaka, Japan), and images were acquired using Multi Gauge software ver. 3.0 (Fujifilm, Greenwood, SC, USA).

The whole-brain samples were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 8000 x g for 10 min. The supernatant from each sample was separated on SDS-PAGE and western blot was performed as described previously 17 (link). Briefly, 10 μg of protein was separated by electrophoresis and transferred to nitrocellulose membranes. Subsequently, the membranes were incubated at 25°C for 1 h with the following primary antibodies; against CRHR-1 (1:1000; GeneTex Inc., Irvine, CA, USA), CRHR-2 (1:1000; Novus Biologicals, Littleton, CO, USA), β-amyloid (1:1000; Rackland Inc., Gilbertsville, PA, USA), Iba (marker of microglia, 1:1000, Wako, Osaka, Japan), IL-6 (1:1000; Abcam, Cambridge, UK), F4/80 (marker of macrophages, 1:1000, Abcam) or β-actin as the loading control (1:5000; Sigma-Aldrich, St. Louis, MO, USA). Next, the membranes were treated with horseradish peroxidase-conjugated secondary antibody (1:1000; Novex, Frederick, MD, USA), and the immune complexes were detected using ImmunoStar Zeta regent (Wako). The images were acquired using the Multi-Gauge software program (Fujifilm, Greenwood, SC, USA).