Anti p16 ink4a

Sections were deparaffinized and rehydrated using three changes of xylenes followed by ethanol gradient. Antigen retrieval was performed using citrate solution pH 6.0 at 60 ℃ overnight, followed by blocking for 1 h using blocking solution (10% goat serum in PBS, 1% tween-20, 1% BSA). Sections were then incubated with primary antibody diluted in DAKO dilution solution (S3022, Agilent, Santa Clara, CA) overnight at 4 degrees then washed and incubated with biotin-conjugated secondary antibody (BP-1100, Vector Biolabs, Burlingham, CA) at room temperature for 2 h. Sections were washed and incubated with streptavidin-HRP using Vectastain ABC-HRP (PK-4000, Vector Laboratories, Burlington, CA) for 20 min. After washing, sections were developed using DAB peroxidase kit (SK4100, Vector Laboratories, Burlingham, CA). Anti-p16-ink4a (PA5-20379, Thermo Fisher), anti-IκB-ζ (NBP-89835, Novus Biologicals, Centennial, CO), anti-3-NT (06-284, Millipore Sigma, Burlington, MA), cleaved caspase-3 (9661 S, CST, Danvers, MA), and Υ-H2AX (2577 S, CST, Danvers, MA) were used in blocking solution (1:100 dilution).

Total protein extracts from cells were run on a 15% sulfate-polyacrylamide electrophoresis gel and transferred to an Immobilon-P transfer membrane (Millipore). The membrane was then blocked with 5% BSA in phosphate-buffered saline, probed overnight at 4°C with appropriate antibodies anti-p16-INK4a (1:500, Invitrogen), anti-p27 Kip1/CDKN1B (1:500, Santa Cruz), anti- γ-H2A.X (1:4000, Abcam), anti-p21 WAF1/CIP1 (1:500, Cell Signaling DGS-60), and PARP (1:1000, Cell Signaling) washed and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G secondary antibody (1:3000). The membrane was incubated with anti-α-tubulin antibodies (1:1000, Santa Cruz) as a loading control. Western blots were run in triplicate. Antibody-labeled protein bands were revealed by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500). The membrane images were analyzed using Image Lab version 5.2.1 (Biorad Laboratories) software.