5-7 μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized with xylene (EMD, Gibbstown, NJ), rehydrated, and H&E stained or processed for immunostaining as previously described (Ramirez et al., 2015 (link)). Anti MSH2 (H-300) rabbit polyclonal antibody (Santa-Cruz) was used at 4°C, overnight or anti K6 rabbit polyclonal antibody (gift from Dr. P. Coulombe, Johns Hopkins University) (Hutton et al., 1998 (link)). Signal was visualized using Alexa-Fluor 488 secondary antibody (Invitrogen, Carlsbad, CA) and mounted with media containing 4’,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) to visualize cell nuclei.
5-7 μm thick frozen tissue sections were fixed in cold acetone (−20°C) for 10 min washed in TBS (Tris-buffered saline) and incubated with 3% H2O2 in methanol for 10 min. Staining was performed as previously described (Ramirez et al., 2015 (link)), using anti-pH2AX Ser139 rabbit mAb (Cell Signaling, Danvers, MA) diluted 1:100 in 3% BSA in TBS with 0.3% triton X-100, Rabbit-on-Farma HRP-Polymer (Biocare Medical) and developed using Betazoid DAB chromogen kit (Biocare Medical). Nikon Eclipse E800 microscope/NIS Elements BR 3.2 software was used for collection of digital images.
5-7 μm thick frozen tissue sections were fixed in cold acetone (−20°C) for 10 min washed in TBS (Tris-buffered saline) and incubated with 3% H2O2 in methanol for 10 min. Staining was performed as previously described (Ramirez et al., 2015 (link)), using anti-pH2AX Ser139 rabbit mAb (Cell Signaling, Danvers, MA) diluted 1:100 in 3% BSA in TBS with 0.3% triton X-100, Rabbit-on-Farma HRP-Polymer (Biocare Medical) and developed using Betazoid DAB chromogen kit (Biocare Medical). Nikon Eclipse E800 microscope/NIS Elements BR 3.2 software was used for collection of digital images.