Anti meox2

FF lung tumor (LT; 3 mm³) samples consisting of almost 70% tumor tissue were pulverized under liquid nitrogen, fixed with 1% formaldehyde for 10 min, and immediately neutralized with 0.125 M glycine for 5 min. The resulting cells were washed with 1X PBS and treated with lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 0.1% sodium deoxycholate, and 0.1% SDS) containing 10 ml with protease inhibitors (complete Mini, Roche, Indianapolis, IN, USA).
The chromatin from the FF-LT samples was sonicated for 10 pulses of 20 seconds w/u 60 watts. Then, 10 μg of chromatin was immunoprecipitated (ChIP) using a commercial EZ-Magna ChIP™ G kit (Millipore, Temecula, CA, USA) and 2.5 μg of anti-MEOX2 (Santa Cruz Biotechnology, Dallas, TX, USA) as well as 1 μg of activated anti-H3K27Ac, anti-H3K4me3, anti-H3K27me3, anti-H3K9me3 or anti-RNA Pol II antibodies (Abcam, Cambridge Science Park, Cambridge, U.K.). A 1-μg aliquot of anti-mouse IgG was used as a negative control for ChIP (Millipore, Temecula, CA, USA).

Immunohistochemical analyses were performed on LNAT, LT and LP tissue samples fixed with formaldehyde 10% or Hope I/II reactive Method (Innovative Diagnostik-System, Germany), and subsequently using 1–2 micrograms of specific antibodies anti-MEOX2, anti-TWIST1, anti-HDAC9, and anti-EVX1 (Santa Cruz Biotechnology, Dallas, Texas, USA), by incubation in a humidity chamber and in addition of the antigen-specific reaction with avidin-biotin peroxidase/diamino-benzidine system (Dako, Carpinteria, CA, USA). A Ventana System device (Roche, Tucson, Arizona, USA) and a transmitted light microscopy study (Leica, Bannockburn, IL, USA), were used to obtain photomicrographs at 40X and 80X magnification.