Anti ki67 antibody

The whole brains of the mice were harvested on day 28 after the allogeneic glioma cells were implanted, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 15 μm thick coronal slices. The sections with the largest tumor area were stained with hematoxylin and eosin or were used for IHC staining.
For IHC analysis, sections were incubated with primary antibodies [1:100 dilution; anti-PPRA antibodies were purchased from Cell Signaling Technology (Proteintech), the anti-Ki67 antibody was purchased from Zsgb Bio (Beijing, China) and the anti-CD34 antibody was purchased from Abcam] overnight at 4 °C, followed by a 1 h incubation at 37 °C with a biotinylated secondary antibody (1:100 dilution). The samples were then incubated with horseradish peroxidase-labeled streptomycoidin and DAB (diaminobenzidine), counterstained with hematoxylin, and visualized using a light microscope.
For the IHC analysis, we quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We assigned the following proportion scores: 1 if 0–25% of the tumor cells showed positive staining, 2 if 26–50% of cells were stained, 3 if 51–75% of the cells were stained, and 4 if 76–100% of the cells were stained; we also divided the different expression levels into four different groups (1 to 4) and scored them.

Liver cancer specimens were collected at the General Hospital of Chinese People's Armed Police Forces. All cases were histologically confirmed to be primary HCC. This study was approved by the Ethics Committee of the General Hospital of the Chinese People's Armed Police Force. All procedures involving human participants were performed in accordance with the ethical standards of the institutional and national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Written informed consent was obtained from all individual participants included in the study.
Immunohistochemical staining was performed as described previously31 on serial 4‐μm thick sections prepared from each tissue block. The primary antibodies used for staining were the anti‐PPP2R3A (Sigma‐Aldrich, 1:200 dilution) and anti‐Ki67 antibody (ZSGB‐BIO, not diluted).
For the Ki67 proliferation assay, five random fields were photographed and the total number of proliferating liver cancer cells is counted using Image‐pro Plus to calculate the positive score for Ki67 staining. The Ki‐67 expression levels were scored according to the criteria of the International Ki67 Working Group.32

Paraffin-embedded tissue sections were used for H&E staining. For IHC analysis, sections were incubated with primary antibodies [1:100 dilution; anti-p-EGFR and anti-p-AKT antibodies were purchased from Cell Signaling Technology (CST), and an anti-Ki67 antibody was purchased from Zsgb Bio (Beijing, China)] overnight at 4 °C, followed by a 1 h incubation at 37 °C with a biotinylated secondary antibody (1:100 dilution). The samples were then incubated with horseradish peroxidase labeled streptomycoidin and DAB (diaminobenzidine), counterstained with hematoxylin, and visualized using a light microscope. RAW 264.7 cells were seeded on 10% collagen-coated glass coverslips and then incubated with cetuximab or nanocapsules for 0, 1, 2 and 4 h. Actin protein was stained by phalloidin. The EGFR protein was labeled with antibodies against EGFR (1:100; CST). Primary antibody labeling was detected by Alexa Fluor 488-conjugated secondary antibodies (Proteintech, Rosemount, IL, USA), followed by confocal imaging with an Olympus FluoView 1200 system (Olympus, Tokyo, Japan). All confocal scanning parameters were held constant among the samples, and the images were minimally processed to maintain the integrity of the data.