Anti cdk2 antibody

Paraffin-embedded samples were cut as 5-μm sections and processed for immunohistochemistry. Tissue sections were prepared for antigen retrieval using microwave treatment in citrate buffer (pH 6.0) and then incubated with anti-NCKAP1 antibody (Proteintech, Rosemont, IL, USA), anti-CDK2 antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-CDK4 antibody (Cell Signaling Technology). Immunostaining was performed using the Envision System with diaminobenzidine as substrate (Dako Cytomation, Glostrup, Denmark). Images were viewed and assessed using an Eclipse 80i microscope (Nikon, Tokyo, Japan). Hematoxylin and eosin (H&E) staining was performed using a Hematoxylin 7211 and Eosin-Y Alcoholic kit (Thermo Fisher Scientific, Inc, Shanghai, China).

Western blot analysis was performed according to previously described procedures¹⁶ . We used primary antibodies, including anti-bile acid receoptor (NR1H4) antibody, anti-SHP antibody (Santa Cruz, CA), anti-phospho-Rb antibody, anti-cyclin D1 antibody, anti-cyclin E1 antibody, anti-CDK2 antibody, anti-CDK4 antibody, anti-CDK6 antibody, anti-p21^Waf1/Cip1 antibody, anti-p27^Kip1 antibody, and anti-β-actin antibody (Cell Signaling Technology) according to the manufacturer’s recommended dilutions.

TMA was constructed using 85 cases of formalin-fixed, paraffin-embedded RCC samples. IHC was performed on TMA to evaluate WTAP and CDK2 protein expression. The tissue samples were stained with the following primary antibodies respectively: anti-WTAP antibody (Abcam, USA) and anti-CDK2 antibody (Cell Signaling Technology, USA). Standard staining protocols were used [24 (link)]. Stained tissues were scored for staining intensity (SI) and the percentage of positive cells (PP). SI was scored on a scale of 0 to 3 (0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining) and PP was scored into five categories: 0 (0% positive cells), 1 (< 10%), 2 (11% to 50%), 3 (51% to 80%) or 4 (> 80%). The final staining score was calculated by multiplying SI and PP score, resulting in a score value ranging from 0 to 12. The positive level of immunohistochemical staining was scored by two urologists and patients with different scores were divided into low- (0–7) and high-staining (8–12) groups.