Quantitative Immunohistochemical Analysis of H3K27Ac in CRC

Staining was performed in 4 μm formalin-fixed, paraffin-embedded tissue sections of CRCs and matched normal mucosa from 10 patients (Additional file 1: Table S5) with the use of Envision Detection System (DAKO). Sections were deparaffinized with xylene and rehydrated in a series of decreasing concentration of ethanol solutions. Heat-induced epitope retrieval was carried out in Target Retrieval Solution (pH 6) (DAKO) in a 96°C water bath, for 20 minutes. After cooling retrieval solutions for 25 minutes at room temperature, the slides were treated for 5 minutes with Blocker of Endogenous Peroxidase (DAKO). Slides were incubated with anti-H3K27Ac (ab4729, Abcam) (diluted 1:500) for 30 minutes at room temperature and subsequently labeled with the Envision Detection System (DAKO). Color reaction product was developed with 3,3′-diaminobenzidine, tetrahydrochloride (DAB) (DAKO) as a substrate, and nuclear contrast was achieved with hematoxylin counterstaining. Representative pictures of each sample were taken at magnification x400 and used for the automatic calculation of the percentage of positively stained nuclear area (labeling index). For this purpose ImmunoRatio i.e. softwere for automated image analysis [37 (link)] was used and the results for normal samples and CRC sections were compared.