Ventana benchmark gx

Resected tissue samples were fixed with 10% formalin, routinely embedded in paraffin, cut into 4 μm thick serial sections, and used for H&E and immunohistochemical staining. Immunohistochemical staining was performed using a Roche Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions. Primary antibodies against p-AKT (#4060, 1:100; Cell Signaling Technology, Danvers, MA, USA), p-mTOR (clone 49F9, 1:100; Cell Signaling Technology), p-S6K1 (#9204, 1:100; Cell Signaling Technology), p-4EBP1 (clone 236B4, 1:500; Cell Signaling Technology), S100 (polyclonal, Ventana Medical Systems), CD31 (clone JC70A, 1:200, Dako), CD34 (clone QBEnd10, 1:200, Dako), D2-40 (760-4395, Ventana Medical System), and PROX1 (ab199359, 1:500; Abcam, Cambridge, UK) were used. Samples were considered positive when at least 10% of the endothelial cells of abnormal vessels exhibited a signal for the targeted protein.

The diagnosis for the deceased patient (F1-IV-2) in Family 1 was confirmed pathologically by a postmortem examination. Immediately after the postmortem examination, organs were fixed in 10% formalin, and 7 µm-thick paraffin-embedded sections were stained with haematoxylin and eosin and Klüver–Barrera staining. Immunohistochemistry was performed with rabbit polyclonal antibody against COL4A1 (1:100, SAB4300825, Sigma-Aldrich, St Louis, MO) and α-smooth muscle actin (1:250, ab7817, Abcam, Cambridge, MA) through the use of a Ventana BenchMark GX automated slide staining system (Ventana Medical Systems, Tucson, AZ) in accordance with the instructions of the manufacturer.

Tissue microarray cores of 2 mm-diameter were obtained from the 305 SNUBH HCCs (SuperBioChips Laboratory, Seoul, Korea). One to three cores from the HCCs and matched non-neoplastic liver were evaluated in this study. Immunohistochemical stains for YAP, TAZ, K19, EpCAM, CAIX, p53, ezrin, urokinase-type plasminogen activator receptor (uPAR), smad2/3 and Ki-67 were performed on 4 μm-thick tissue microarray sections manually or by using the Ventana BenchMark GX automated platform (Ventana Medical Systems). The details for each antibody are summarized in Table 1.
The presence of cytoplasmic expression in >5% of the tumor cells was regarded as positive for K19, uPAR and smad2/3 expression. EpCAM, CAIX and ezrin were expressed in the tumor cell membranes. Although YAP and TAZ were expressed in both the cytoplasm and nuclei, only unequivocal nuclear or nucleocytoplasmic staining for YAP and TAZ were interpreted as positive. Cytoplasmic YAP/TAZ expression without the nuclear stain was not counted as positive regardless of the intensity. Strong nuclear p53 expression in >5% of tumor cells was interpreted as p53 overexpression. The Ki-67 labeling index was calculated as the percentage of Ki-67-stained tumor cell nuclei over the total number of nuclei in a ×400 magnification field, with the use of ImageJ analysis software (downloaded from imagej.nih.gov/ij, version 1.47).

The colorectal tissue sections (5 μm thick) were deparaffinized, rehydrated, rinsed with ddH₂O, and washed with Tris-buffered saline (TBS). IHC staining was carried out using an automated Ventana BenchMark GX instrument (Ventana Medical Systems, Inc., Tucson, AZ, USA). Probesof anti-MeCP2 (3456, Cell Signaling Technology) and anti-pMeCP2 antibody (AP3693a, Abgent), and a 5mC antibody (AMM99021, AVIVA, San Diego, CA, USA) was used. The intensity of staining was determined using ImageJ1.50i. Randomly selected 10 microscopic fields were subjected to semi-quantification from computer assisted image analyses.

Serial 5 μm sections were taken from the paraffin blocks to contain the same cell with the microtome (Leica RM2145). Five-micron sections were taken from the whole tissue block onto positively charged slides. Slides were kept under suitable conditions for hematoxylin and eosin staining and immunohistochemical (IHC) staining. At the end of the deparaffinization process, 5-micron sections were taken on a positively charged slide, and histochemical staining was performed with caspase-3 (Santa Cruz Bio., China) and nuclear factor kappa B (NF-κB) (Santa Cruz Bio., China) antibody in Ventana Benchmark GX automatic immunohistochemistry stainer.
Light and IHC Staining Preparations Olympus BH 40 brand light microscope with camera attachment was photographed and interpreted. The sections examined under the light microscope were scored as none (−), mild (+), moderate (++), and severe (+++).

Resected tissue samples were fixed with 10% formalin, embedded in paraffin, cut into 4-µm-thick serial sections, and used for hematoxylin and eosin (H&E) and immunohistochemical staining. TNM classification was performed according to the 8th edition of the Union for International Cancer Control. Immunohistochemical staining with a primary antibody against p53 (clone-DO7) and p16 (clone-E6H4) was performed using a Roche Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s instructions.