Horseradish peroxidase labeled goat anti mouse igg h l

Total protein from each sample from retinas was extracted using radioimmunoprecipitation assay (RIPA) buffer (Solarbio Science, Beijing, China). The bicinchoninic acid (BCA) method was used to determine the protein concentrations (ThermoFisher). A total of 50 μg of each sample was electrophoresed (80 V) and subsequently transferred (at 4°C, 350 mA) to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies [p-PI3K (1:2000; mouse; Cat# sc-12929; Santa Cruz Biotechnology Inc.), phospho-Akt 1/2/3 (1:2000; mouse; Cat# sc-101629; Santa Cruz Biotechnology Inc.), and vascular endothelial growth factor (1:2000; mouse; Cat# sc-365578; Santa Cruz Biotechnology Inc.)] for 16 hours at 4°C after blocking with 5% non-fat milk. The membranes were then incubated with horseradish peroxidase-labeled goat anti-mouse IgG(H+L) (1:2000; Cat# ZB-5305; Zhongshan Jinqiao Biotechnology Co. Ltd.) for 1 hour at room temperature. Chemiluminescence reagents (Millipore, Waltham, MA, USA) and an imaging system (GE AI680, Boston, MA, USA) were used to visualize the bands. ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to calculate gray value.

Immunohistochemistry was performed using a Streptavidin Biotin Complex immunohistochemistry kit (Boster Bioengineering Co., Wuhan, China). The paraffin sections were dewaxed and antigen was repaired and sealed with goat serum. Then the sections were incubated overnight at 4°C with primary antibodies [phospho-PI3K (p-PI3K; 1:2000; mouse; Cat# sc-12929; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), phospho-Akt 1/2/3 (1:2000; mouse; Cat# sc-101629; Santa Cruz Biotechnology Inc.), and vascular endothelial growth factor (1:2000; mouse; Cat# sc-365578; Santa Cruz Biotechnology Inc.)], followed by incubation at 37°C for 30 minutes with horseradish peroxidase-labeled goat anti-mouse IgG(H+L) (1:2000; Cat# ZB-5305; Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China) on the next day. After 3,3’-diaminobenzidine staining and hematoxylin staining, photographs were taken under the light microscope.