Blocker of endogenous peroxidase

Immunohistochemical staining (IHC) was applied to visualize TP53 expression level in 112 tumors that were included in TP53 coding sequence analysis. Envision Detection System (DAKO, Glostrup, Denmark) was used for staining procedure according to manufacturer’s recommendations. Four-μm FFPE tissue sections were deparaffinized with xylene and rehydrated in a series of ethanol solutions of decreasing concentration. Heat-induced epitope retrieval was achieved by 30 minutes incubations of the samples in Target Retrieval Solution pH 9 (DAKO) in a 96°C water bath. The slides were treated with a Blocker of Endogenous Peroxidase (DAKO) for 5 minutes followed by incubation with the primary monoclonal mouse anti-human TP53 antibody (clone DO-7, ready to use concentration, DAKO) for 1h. Diaminobenzidine tetrahydrochloride (Dako) was used as substrate to visualize immunoreactivity followed by hematoxylin nuclear counterstaining. Analysis of nuclear immunohistochemical reactivity was performed by calculating H-score, that combines information on both reaction intensity (scored from 0 to 3) and number of the cells with a given intensity. Previously reported formula was used for quantification (17 ). Three high power fields (magnification x400) were evaluated for each sample. Scoring results were analyzed as continuous variables.

Immunohistochemical staining (IHC) was performed on 4 μm FFPE tissue sections using the Envision Detection System (no. K500711-2, DAKO, Glostrup, Denmark). Sections were deparaffinized with xylene and rehydrated in a series of ethanol solutions of decreasing concentration. Heat-induced epitope retrieval was carried out in a Target Retrieval Solution pH 9 (DAKO) in a 96 °C water bath, for 30 min. Cooled slides were treated with a blocker of endogenous peroxidase (DAKO) for 5 min and subsequently incubated with the primary antibodies as indicated in Table 2. The color reaction product was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAKO) as a substrate, and nuclear contrast was achieved by hematoxylin counterstaining. Analysis of immunohistochemical reactivity was performed by calculating the H-score, which combines information on both reaction intensity (scored from 0 to 3) and the number of the cells with a given intensity. A previously reported formula was used for quantification [16 ]. Scoring results were analyzed as continuous variables. For each SCA sample, immunostaining against T-PIT was performed to verify the corticotroph nature of the tumors.

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.