Hrp conjugated goat anti rabbit igg secondary antibody

The tissues were vacuum inflation-fixed and paraffin embedded, and 5-μm sections were cut. Sections were used for immunohistochemical examination. Immunohistochemical staining was performed with the following primary antibodies: rabbit monoclonal anti-LC3 (Cell Signaling Technology), rabbit recombinant monoclonal anti-p62 (Bimake), rabbit monoclonal anti-FN (Abcam), and rabbit polyclonal anti-GLP-1R (Bioss) followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody according to the kit instructions (ZSGB-BIO, Beijing, China). Peroxidase conjugates were subsequently visualized by using diaminobenzidine (DAB) solution. The sections were then counterstained with hematoxylin and mounted on cover slips. Between each step, the cells were extensively rinsed three times for 5 min each time. HE and Masson staining were performed according to the manufacturer’s instructions. The staining kits were purchased from Nanjing JianCheng Bioengineering Institute (Nanjing, China). Staining was photographed using a Leica microscope (DM2500, Wetzlar, Hesse, Germany). For each rat, five fields were chosen to obtain the average of integrated optical density (iod) of IHC staining and the average collagen area indicated by Masson staining by Image Pro Plus (IPP) software. Average areas from 5 rats were determined, and the analysis was done blindly.

Expressions of NGF, vascular endothelial growth factor (VEGF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by WB analysis. Proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Haimen, China). Protein concentration was quantified with a bicinchoninic acid (BCA) protein assay kit (Beyotime). From each extract, 80 µg total protein was separated with 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% nonfat milk at room temperature for 1 hr and incubated overnight at 4°C with rabbit anti-VEGF (VEGF; 1∶1,000, Santa Cruz, CA, USA), anti-NGF (1∶1500, Epitomics, Burlingame, CA), or anti-GAPDH antibody (1∶3,000; CoWin Bioscience, Beijing, China). The next day, blots were incubated with appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1∶10,000, ZSGB-BIO, Beijing, China). Blots were developed using an enhanced chemiluminescence (ECL) detection kit (Millipore) and visualized using a FluroChem E Imager (ProteinSimple, Santa Clara, CA, USA). Then, the protein expression levels were quantified with Quantity AlphaEaseFCTM (Alpha Innotech, San Leandro, CA, USA) imaging software. Relative expression of target proteins was normalized to GAPDH.

Glycogen synthase (GYS) expression levels were assessed by IHC staining. Liver slices were incubated with anti-GYS primary antibody (Cat. 3886, Cell Signaling Technology) at 4 °C overnight, then incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat. PV-6000, ZSGB-Bio, Beijing, China) for 20 min and stained with 3,3 N-Diaminobenzidine tertrahydrochloride (DAB) and counterstained with hematoxylin.