Phospho yh2ax

Keratinocytes were differentiated via organotypic raft culture as described previously (62 (link), 76 (link), 79 (link)). Briefly, cells were seeded onto type 1 collagen matrices containing J2 3T3 fibroblast feeder cells. Cells were cultured to confluence atop the collagen plugs, lifted onto wire grids and cultured in cell culture dishes at the air-liquid interface. Media was replaced on alternating days. Following 14 days of culture, rafted samples were fixed with formaldehyde (4% vol/vol) and embedded in paraffin. Multiple 4 μm sections were cut from each sample. Sections were stained with hematoxylin and eosin (H&E) and others prepared for immunofluorescent staining via HIER. Fixing and embedding services in support of the research project were generated by the VCU Massey Cancer Center Cancer Mouse Model Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059. Fixed sections were antigen retrieved in citrate buffer and probed with the following antibodies for immunofluorescent anaylsis: phospho-yH2AX 1/500 (Cell Signaling Technology; 9718), Involucrin 1/1000 (abcam; ab27495), and Keratin 10 1/1000 (SigmaAldrich; SAB4501656). Cellular DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI, Santa Cruz sc-3598). Microscopy was performed using the Keyence imaging system,

Cells were grown on coverslips to 70% confluence, fixed with formaldehyde, and permeabilized with NP-40. Cells were incubated with the primary antibody for 1 h (phospho-yH2.AX; Cell Signaling Technology 20E3; 1/500), diluted in 10% normal goat serum. Coverslips were washed three times in PBS-Tween (0.1% Tween), and immune complexes were visualized using Alexa 488- or Alexa 595-labeled anti-species-specific antibody conjugates (Molecular Probes). Cellular DNA was stained by inclusion of 4′,6-diamidino-2-phenylindole (DAPI; Santa Cruz sc-3598) in the penultimate wash. Microscopy was performed at the VCU Microscopy Facility. Immunofluorescence was observed using an LSM 700 laser scanning microscope and ZEN 2011 software (Carl Zeiss). Images were assembled in Adobe Photoshop CS 6.0.