Bond 3 automated ihc ish stainer

The FFPE were cut into 4 μ sections, pretreated in EDTA-buffer at pH 9.0 for 20 min and incubated with mouse monoclonal antibodies against BAP-1 (clone C-4, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:40. The dilution had been gradually titrated until optimal staining was achieved, according to manual control by a board certified pathologist (GS). A red chromogen was used. Finally, the sections were counterstained with hematoxylin and rinsed with deionized water. The deparaffinization, pretreatment and staining steps were run in a Bond III automated IHC/ISH stainer (Leica, Wetzlar, Germany).

Livers were processed as mentioned above (H&E staining section). Following dewaxing and antigen retrieval, liver sections were stained with a BOND Polymer Refine Detection Kit (DS9800, Leica) by BOND-III Automated IHC/ISH Stainer (Leica). Ki67 staining was examined by light microscopy.

TMAs were acquired from the Mid-Atlantic Division of the Cooperative Human Tissue Network-based at the University of Virginia containing a total of 398 stage II invasive BC cores, as well as 20 normal breast tissue cores, 12 breast fibroadenoma cores, 5 breast cell line controls (MCF-7, MCF-10a, SK-BR-3, SK-OV-3, T-47D) and 5 non-breast controls (salivary gland, endometrium, colon, tonsil, prostate). After exclusion of damaged sections and cases with missing information, 316 cases were included. The following optimized staining conditions were chosen: monoclonal mouse anti-human FOXM1 (A-11, SCBT) at 1:120 dilution, and monoclonal mouse anti-human CBP (C-20, SCBT) at 1:100. Staining was performed on 4 slides per antibody using a BOND-III Automated IHC/ISH Stainer (Leica, Buffalo Grove, IL, USA) by the USC Clinical Immunohistochemistry laboratory. A three-tier scoring system was devised: 0-no staining, 1-weakly positive, 2-strongly positive. Only tumors with a score 2 were included in the subsequent analysis. Clinical variables used for TMA analysis are listed in Table S24.

Livers were processed as mentioned above (H&E staining section). Following dewaxing and antigen retrieval, liver sections were stained with a BOND Polymer Refine Detection Kit (DS9800, Leica, Munich, Germany) by BOND-III Automated IHC/ISH Stainer (Leica, Germany). Gp130 and Ki67 staining was examined by light microscopy. Positive cells were quantified from 3 randomly selected 200× field images of liver sections (saline: n = 5 mice/genotype; APAP: n = 6 mice/genotype) using the cell counter function in ImageJ software (National Institute of Health, Bethesda, MD, USA).

Paraffin blocks were cut into 4 μm sections, pretreated in EDTA-buffer at pH 9.0 for 20 min and incubated with mouse monoclonal antibodies against BAP-1 (clone C-4, catalog no. sc.28383, Santa Cruz Biotechnology, Dallas, Texas, USA), IDO (catalog no. 05‐840, Sigma‐Aldrich, Saint Louis, MO, USA), and rabbit monoclonal antibodies against IGF-1R (catalog no. ab39398, Abcam, Cambridge, UK), and TIGIT (catalog no. ab233404, Abcam). A red chromogen was used, and sections were counterstained with hematoxylin and rinsed with deionized water. The deparaffinization, pretreatment, primary staining, secondary staining and counterstaining steps were run in a Bond III automated IHC/ISH stainer (Leica, Wetzlar, Germany). Dilutions between 1:20 and 1:1500 had been evaluated by a pathologist (GS), before selecting 1:40 for BAP-1, 1:1000 for IGF-1R and 1:75 for IDO and TIGIT. For assessment of BAP-1 status in a light microscope, we followed a previously used classification^{7 (link),9 (link)}. The area in a lesion with the most intense nuclear BAP-1 staining was selected under low-power magnification (×40). The percentage of melanocytes with any visible immunoreactivity above background was then assessed in 3 high-power fields (×200). Lesions with ≥33% immunoreactive cells were classified as BAP-1 positive, and lesions with <33% immunoreactive cells as BAP-1 negative.

Procedures examining the expression of p16 protein were carried out using a Bond-III Automated IHC/ISH Stainer (Leica Biosystem, Wetzlar, Germany) according to manufacturer’s instruction and reagents. Briefly, where available, 4 μm FFPE sections were mounted on glass microscope slides coated with Poly-L-Lysine. They were deparaffinized using Bond Dewax Solution (Leica Biosystem), rehydrated, and washed with Bond Wash Solution. The slides were incubated with Bond Epitope Retrieval Solution and heated at 100 °C for 20 min, washed, and Peroxide Wash Solution applied for 5 min. The p16 primary antibody (mouse monoclonal Anti-p16INK4a (E6H4), Ventana, Tucson, AZ, USA) was added on the slides for 15 min, followed by the anti-mouse secondary antibody (Post Primary Rabbit anti mouse IgG, ProClin, Leica Biosystem) for 8 min and the Bond Polymer Refine Detection solutions with intermittent washing. Slides were counterstained with Hematoxylin, and then dehydrated and mounted with DPX by using a Tissue-Tek film coverslipper (Sakura Finetek, Tokyo, Japan). Negative controls were obtained by excluding the primary antibody. Scoring of p16 IHC cytoplasmic and nucleic staining were evaluated by an experienced pathologist, based on defined characteristics whereby p16 was scored as positive if it was strong and diffuse (>70% of tumor cells), and negative if absent, weak, or focal [50 (link)].