Anti ki67

During the administration period, the body weight of the nude mice was measured every 4 days, and the longest diameter (a) and shortest diameter (b) of the tumor were measured with a Vernier caliper. Accordingly, 48 h after the last administration, the experimental animals were euthanized, the tumor was weighed, and the tumor inhibition rate was calculated. The tumor tissues were fixed in a 10% formaldehyde solution for 24h and were subsequently fixed in formalin and embedded in paraffin. Sections of the dissected biopsies were stained with hematoxylin and eosin (H&E) using standard procedure and were examined under light microscopy. Paraffin-embedded sections were stained with anti-PCNA (Abcam, USA), anti-Ki-67 (Maixin, China), and anti-Cleaved-Caspase3 monoclonal antibody (CST, USA). HRP-conjugated anti-mouse secondary antibody was added and detected according to the manufacturer’s protocol (Maixin, China). Moreover, the imageJ software was used for image analysis.

Formalin-fixed and paraffin-embedded sections (4 μm) were obtained for immunohistochemical staining according to the standard protocol. Briefly, deparaffinized and rehydrated sections were treated with 3% H₂O₂ and subjected to antigen retrieval by citrate buffer (pH 6.0). Then, sections were blocked with 5% BSA for 20 minutes and incubated overnight (16–18 h) with primary antibody (Anti-Ribonuclease T2 antibody, 1:100, ab107313, anti-TM4SF1, 1:200, ab113504, and anti-S100A12, 1:250, ab37657, Abcam, Cambridge, MA, USA; anti-NF-κB p65, 1:100, sc-109, Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-ERK, 1:250, #4695, Cell Signaling Technology, Beverly, MA, USA; Anti-CD34 [MAB-0034, QBEnd/10], anti-Ki67 [MAB-0129, MIB-1], and anti-p53 [MAB-0142, DO-7], Ready-to-use; Maixin Bio, Fujian, China) at 4°C. Then they were incubated with biotinylated linked antibodies and peroxidase-labeled streptavidin (UltraSensitive^TM SP (Mouse/Rabbit) IHC Kit-9710; Maixin Bio, Fujian, China) for 15 min each at room temperature. The reaction products were stained with 3,3′Diaminobenzidine (DAB) and slight counterstained with hematoxylin. The sections with PBS, replacing the primary antibody, and served as negative control.