Goat anti mouse igg

Ten-μm thick frozen muscle cross-sections were cut from the mid-belly of each muscle at −20 °C with a cryostat (Leica, Wetzlar, CM1850, Germany), and stored at −80 °C for further staining. Immunohistochemistry was used to determine muscle fiber cross-sectional area and fiber type composition. The sections were air dried for 10 min and fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 min. Then sections were incubated in a blocking solution (5% BSA) (Boster, Wuhan, China) for 30 min at room temperature and in turn incubated in a primary antibody solution at 4 °C overnight. The primary antibodies included an anti-skeletal fast myosin antibody (Sigma, St. Louis, MO) to visualize the type II myosin heavy-chain (MHC) in SOL muscle, and an anti-skeletal slow myosin antibody (Sigma) to visualize the type I MHC in both GAS and EDL muscles. Subsequently, sections were placed in goat anti-mouse IgG (Boster) for 30 min, in SABC (Boster) for 30 min and in DAB (Boster) for 5–15 min at room temperature. These sections were viewed and captured as digital images using a VHX-5000 Digital Microscope (KEYENCE Corporation, Osaka, Japan) at an objective magnification of 40×. A minimum of 3 fibers or 600 cells was counted in each sample.

Immediately after the rats were euthanized (Figure 1B), their mPFC, CeA, and hippocampal CA1 were retrieved immediately through dissection and stored at –80°C until analysis (Li et al., 2016 (link), 2017 (link)). The tissues were homogenized and lysed using a radio-immuno-precipitation assay buffer (AR0102, Boster) and then subjected to the measurement of protein concentration using the bicinchoninic acid kit (AR1189, Boster). The lysates (20 μg each) were separated on 10% SDS-PAGE gels, and the separated protein bands were transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane. Immunoreactivity was determined based on enhanced chemiluminescence, and the signals were detected using a Bio-Rad ChemiDoc system (Bio-Rad Laboratories, China).
The following antibodies were used: anti-GAPDH (1:5,000, BM1623, Boster); anti-GluA1 (1:1,000, A1826, ABclonal); anti-mGluR1 (1:1,000, abx112750, Abbexa); anti-mGluR5 (1:1,000, A3758, ABclonal); anti-GluN2B (1:1,000, abx23583, Abbexa); anti-PSD-95 (1:1,000, abx236850, Abbexa); anti-α5 GABA (1:1,000, ab259880, Abcam). The secondary antibodies used were goat anti-rabbit IgG (1:10,000; Boster) and goat anti-mouse IgG (1: 10,000, Boster). The immunosignals were quantified using densitometry and were expressed relative to the GAPDH signals and normalized to the control for data analysis.

Assay kits for malondialdehyde (MDA, cat: 20180613), total superoxide dismutase (T-SOD, cat: 20180616), glutathione (GSH, cat: 20180615), catalase (CAT, cat: 20180613), total protein (TP, cat: 20180610), alkaline phosphatase (ALP, cat: 20180609), AST (cat: 20180608), ALT (cat: 20180607), globulin (GLB, cat: 20180606), total bile acid (TBA, cat: 20180605), and cholesterol (CHOL, cat: 20180604) were purchased from Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Mouse anti-β-actin (cat: 66240-1-lg), goat anti-mouse IgG (cat: BST12F21C50), rabbit anti-heme oxygenase-1 (HO-1, cat: ZP1039BP39), goat anti-rabbit IgG (cat: BST12L05A54), rabbit antitumor necrosis factor-α (TNF-α) (cat: BST12F21C48), mouse anti-intercellular cell adhesion molecule-1 (ICAM-1) (cat: BST12F21C50), and immediate SABC-POD (goat anti-rabbit IgG, cat: 12H25C) kit were purchased from Boster Biological Technology Co., Ltd. (Wuhan, China). Rabbit anti-nuclear factor erythroid-2 related factor 2 (Nrf2) (cat: 16396-1-AP) and rabbit anti-β-tubulin (cat: 16385-1-AP) antibodies were obtained from Proteintech Co., Ltd. (Chicago, IL, USA).

Western blot analysis was performed as described previously (22 (link)). Briefly, cytoplasmic and nuclear proteins were isolated using the Nucleus and Cytoplasm Protein Extraction Kit (Beyotime, P0028, China). In some cases, cells were lysed with lysis buffer (Life technologies, 87788, USA). Cell extracts were subjected to 10 or 15% SDS-PAGE and transferred onto PVDF membranes (Millipore 0.45 μm or 0.22 μm) followed by blocking with 5% non-fat milk in Tris-buffered saline-Tween (TBST, 50mM Tris-HCl pH 7.5, 200mM NaCl, 0.1% (v/v) Tween-20) at room temperature for 2 h. The membrane was clipped according to the molecular weight of the protein, and then probed with an appropriate primary antibody at room temperature for 2 h. After three washes with TBST, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (BOSTER, BA1054, China), goat anti-mouse IgG (BOSTER, BA1051, China) or donkey anti-goat IgG (Beyotime, A0181, China) at room temperature for 1 h. Protein bands were visualized by exposure to FluorChem HD2 Imaging System (Alpha Innotech) after the addition of chemiluminescent substrate (Beyotime, P0018, China). Protein molecular weight markers were purchased from Thermo Fisher (26616, USA) and YEASEN (20352, China).

EPC cells cultured in six-well plates were transfected with 2 µg plasmid. Twenty-four hours after transfection, cells were lysed by the RIPA (radio immunoprecipitation assay) lysis buffer from beyotime. Then western blot was performed as previously described (Mo et al., 2010 (link)) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as loading control. The primary antibodies used for western blot were rabbit anti-Firefly Luciferase (Abcam, #ab185923, 1:5000) and mouse anti-GAPDH (Boster, #BM3876, 1:100). The secondary antibodies were goat anti- rabbit IgG (immunoglobulin) (Boster, #BM3894, 1:5000) and goat anti-mouse IgG (#BM3895, 1:5000; Bosterbio, Pleasanton, CA, USA), respectively. Intensity of the bands in the western blot image was analyzed by the ImageJ software (http://rsb.info.nih.gov/ij/).

Cells and tissues were lysed on ice in RIPA lysis buffer supplemented with protease inhibitor (Beyotime, Shanghai, China). Protein expressions was detected by western blot analysis as previously described [25 ]. The following primary antibodies were used: anti-E-cadherin, anti-N-cadherin, anti-vimentin, anti-Met, anti-pMet (Try1003, Try1234/1235 and Try1349), anti-Akt, anti-pAkt, anti-GSK-3β and anti-pGSK-3β (Cell Signaling Technology, Danvers, MA); anti-fibronectin and anti-ZEB2 (Abcam, Cambrige, MA); anti-MACC1 (Abnova, Taipei, China), β-actin (Boster, Wuhan, China) and HRP-conjugated anti-GAPDH (Kangchen, Shanghai, China). Protein signals were developed after incubation with HPR-conjugated goat-anti-rabbit IgG (SouthernBiotech, Birmingham, AL) or goat-anti-mouse IgG (Boster) secondary antibody by using the chemiluminescence (ECL) system. The expression level of MACC1 protein in the GC tissues was half-quantified with Image J software.