B201 optical microscope

We used a biotin-streptavidin HRP detection system (SP-9000; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, People’s Republic of China) to perform immunohistochemistry. Formalin-fixed, paraffin-embedded eye tissue sections (3.5 μm thick) were placed on slides, deparaffinized in xylene, and rehydrated by incubation in graded ethanol baths in PBS. Endogenous peroxidase was blocked with hydrogen peroxide. The sections were then treated with goat serum for 30 min and incubated overnight with the following antibodies: rabbit anti-Smo polyclonal antibody (1:50 dilution; ab72130; Abcam, Cambridge, UK), rabbit anti-Gli1 polyclonal antibody (1:50 dilution; sc-20687; Santa, Santa Cruz, CA, USA), mouse anti-VEGF monoclonal antibody (1:100 dilution; sc-7269; Santa, USA) at 4°C. Subsequently, they were incubated with Biotin-labeled goat anti-rabbit/mouse IgG secondary antibodies (SP-9000; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, People’s Republic of China) for 15 min at room temperature. The primary antibody was replaced with PBS for the negative controls, and 3,3′-diaminobenzidine was used as the chromogen. The images were observed and captured using an Olympus B201 optical microscope (Olympus, Tokyo, Japan).

Formalin-fixed, paraffin-embedded 6 μm eye tissue sections were placed on slides, deparaffinized in xylene, and rehydrated in graded ethanol baths in phosphate-buffered saline. Immunostaining was performed by the streptavidin-peroxidase method (Ultrasensitive; MaiXin, Fuzhou, People’s Republic of China). Hydrogen peroxide (3%) was applied to block endogenous peroxidase activity, and normal goat serum was used to reduce nonspecific binding.
Sections were incubated with commercially available primary antibodies: rabbit anti-CCN1 polyclonal antibody (1:500, Abcam, Cambridge, UK); rabbit anti-p-AKT1/2/3 (Ser473) polyclonal antibody (1:500, Santa Cruz Biotechnology Inc., Dallas, TX, USA); or rabbit anti-VEGF polyclonal antibody (1:500, Santa Cruz Biotechnology Inc.) overnight at 4°C. The sections were incubated with biotinylated secondary antibody (1:1,000, Santa Cruz Biotechnology Inc.) and reacted with the avidin-biotinylated peroxidase complex. The primary antibody was replaced with phosphate-buffered saline for negative controls. The peroxidase reaction was developed with 3, 3′-diaminobenzidine (Maixin Biotechnology, Foshan, China), and sections were counterstained with hematoxylin, dehydrated with alcohol, and mounted using a standard procedure. Images were digitally captured using an Olympus B201 optical microscope.