Anti p21cip1 sc 397

Tumors generated in mice were dissected, fixed in 4% formalin for 48 h and embedded in paraffin. Four-micrometer thick sections were deparaffinized, rehydrated and heated for 10 min in citrate buffer for antigen retrieval. Endogenous peroxidase was blocked with 5% hydrogen peroxide in methanol for 15 min. After incubation with blocking solution, sections were incubated with the respective primary antibody, anti-SOX2 (ab97959 Abcam, Cambridge, UK), anti-Ki67 (ab15580, Abcam, Cambridge, UK), anti-p21^CIP1 (sc-397 Santa Cruz, Dallas, TX, USA) and anti-p27^KIP1 (sc-1641 Santa Cruz, Dallas, TX, USA), at 37 °C for 2 h. The sections were then washed and incubated with MACH 3 Rabbit Probe and MACH 3 Rabbit HRP-Polymer (M3R531, Biocare Medical, Pacheco, CA, USA). Color was developed with 3,3′-Diaminobenzidine (DAB) and nuclei were counterstained with hematoxylin.

HL-derived L428 and KMH2 cells were grown in suspension in RPMI 1640 supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (FCS), in a 5% CO2 humidified atmosphere at 37 °C. For the induction of senescence cells were treated with 50 μM H₂O₂ for 2 h in RPMI without FCS, washed with complete medium and grown for 4–7 days.
Antibodies used included: anti-p21 ^Cip1 (sc-397), anti-p16 ^INK4a (sc-1207), anti-p53 (sc-126) and anti-p65 (sc-8008) and phospho-NF-κB p65 (Ser 536) (sc-33020- R) which were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-γH2AX phosphor -S139 (ab18311, Abcam, Cambridge, MA, USA), anti-trimethyl Histone H3 (Lys4/Lys9) (Millipore 07-992). Anti CD15 LeuM1 and anti-CD30 BerH2 (Dako, Copenhagen, Denmark), Ki-67, (ab15580, Abcam). For florescence detection the following secondary antibodies were used: donkey anti-rabbit IgG (DyLight 549, 711-505-152, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Alexa flour-488 conjugated goat anti-mouse (Molecular Probes Inc., Eugene, OR, USA).