Microscope vs120 whole slide scanner

Tumor tissues were fixed with neutral buffered 10% formalin solution (HT501128, Sigma), embedded in paraffin, cut into sections at 4 μm, and deparaffinized. Antigen retrieval was performed using sodium citrate buffer (10 nM sodium and 0.05% Tween 20 at pH 6.0) at 96^oC for 30 minutes. Sections were incubated with anti-Ki67 (1:50, A2094, Abclonal) or PPP1R12A (1:50, 22117010AP, proteintech, China) for 120 minutes at room temperature. Primary antibodies binding to tissue sections was visualized using DAB Detection kit (ZLI-9017, Zsbio, China), and counterstained with hematoxylin. All images were captured using a Microscope VS120 Whole Slide Scanner (Olympus) and analyzed using the computer-assisted Image-Pro Plus software (Meida Cybernetics, Bethesda, MD). The quantification of Ki67, and PPP1R12A staining was performed as positive area percent. Data were analyzed in a blinded fashion by two independent observers.

The tissue paraffin sections were heated in an oven at 60 °C for 2 h, followed by dewaxed with xylene for 20 min, and dehydrated with gradient ethanol solution. Then sections were washed with PBS three times, incubated in 3% hydrogen peroxide for 10 min, washed with PBS three times again, and put in sodium citrate buffer (10 nM sodium and 0.05% Tween 20 at pH 6.0) at 96 °C for 30 minutes for antigen repair. After being blocked with calf serum for 20 min, primary antibodies were added and incubated for 60 minutes at room temperature, including Ki67 (1: 50, A2094, Abclonal), ZEB1 (1:50, 21544-1-AP, Proteintech, China), E-cadherin (1:500, 20874-1-AP, Proteintech, China) and vimentin (1:1000, 3932, CST, USA). After rinsed with PBS three times, the secondary antibodies were incubated at 37 ° C for 30 min, washed with PBS three times, and developed with DAB (ZLI-9017, Zsbio, China). Sections were then counterstained with hematoxylin, dehydrated with ethanol, cleared with xylene, and mounted. All images were captured using a Microscope VS120 Whole Slide Scanner (Olympus) and analyzed using the computer-assisted Image-Pro Plus software (Meida Cybernetics, Bethesda, MD). The quantifications of Ki67, and ZEB1, E-cadherin and vimentin staining were performed as positive area percent. All specimens were confirmed by two independent observers.