Bond max automated immunostainer

Sections (4 μm thick) were obtained from each TMA block, and we performed deparaffinization and rehydration with routine techniques (e.g., immersing in xylene and graded ethanol). Then, antigen retrieval (heat-induced, 100 °C for 20 min in sodium citrate buffer) and blocking of endogenous peroxidase activity (for 15 min in S2023 peroxidase-blocking solution, Dako, Glostrup, Denmark) were performed. For IHC staining of DUSP4, rabbit anti-DUSP4 polyclonal antibody (1:200, ab72593, Abcam, Cambridge, MA, USA) was used. Detection was achieved using an EnVision Detection System (K5007, Dako, Glostrup, Denmark). A series of IHC staining procedures were performed according to the manufacturer’s instructions using a Bond Max Automated Immunostainer (Leica Biosystems, Nussloch, Germany). DUSP4 expression was defined as nuclear staining of tumor cells. We classified each case as DUSP4 negative (lack of staining or staining in <10%) or DUSP4 positive (staining in ≥10%). There is no standard cutoff for DUSP4 expression in RCC. However, it is common to set a cutoff of 10% for novel biomarkers [17 (link),18 (link)]. The staining intensity was not assessed in this study due to concerns of subjectivity and reproducibility. Two pathologists (Bang, S. and Paik, S.S.) evaluated DUSP4 expression without consideration of clinicopathological data.

Immunohistochemical studies were carried out on formalin-fixed, paraffin-embedded, 4-µm-thick tissue sections. Immunostaining for Ki-67 was performed with a BOND-MAX™ automated immunostainer (Leica Biosystems, Melbourne, Australia) and the BOND™ Polymer refined detection kit (Vision Biosystems, Melbourne, Australia). Briefly, antigen retrieval was performed at 97 °C for 20 min in ER1 buffer. After blocking endogenous peroxidase activity with 3% hydrogen peroxidase for 10 min, primary antibody incubation was performed for 15 min at room temperature using an antibody dilution of 1:200. Apoptotic cells were analyzed by TUNEL assay using the DeadEnd colormetric TUNEL assay kit (Promega, Madison, WI) as previously described.

Formalin-fixed samples were embedded in paraffin, following standardized protocols obtained from routine diagnostic practice. From each sample, serial 4-μm-thick sections were stained with hematoxylin and eosin and submitted for immunohistochemical analysis. The latter was performed in the BOND-MAX automated immunostainer (Leica Biosystems, Wetzlar, Germany) using the anti-CD20 primary antibody (clone L26, dilution 1:50; Dako, Agilent, Santa Clara, USA). Histological sections were jointly examined by two expert hematopathologists who were blinded to the experimental conditions. Morphometric analyses were performed manually, using the Leica DFC295 digital color camera and LAS X software (Leica Microsystems, Wetzlar, Germany).

Formalin-fixed and paraffin-embedded sections of TMA were deparaffinized in xylene and were rehydrated with gradually decreasing concentrations of alcohol. Sections were immunostained after antigen retrieval using the Bond-max automated immunostainer (Leica Microsystems, Newcastle, UK). Primary antibodies used were the anti-CD8 polyclonal antibody (1:100, Neomarkers, Fremont, CA, USA), the anti-CD45RO monoclonal antibody (1:50, Neomarkers) and the anti-FOXP3 monoclonal antibody (1:50, Abcam, Cambridge, MA, USA). Antibody binding was detected by using the Bond Polymer Refine Detection kit (Leica Microsystems).
After immunohistochemical staining for each of the T cell markers, TMA slides were scanned by the Aperio image analysis system (Leica Biosystems, New Castle, UK) (Fig 2). The software counted the number of immunopositive nuclei in each tissue core. The average density (cells/mm²) of each lymphocyte subset was calculated in a whole TMA core. T cell subset densities were divided into two groups (high versus low) according to a median-split.

Two micrometre thick temporal artery sections from FFPE samples were used for immunohistochemistry. After 20-minute antigen retrieval with citrate buffer (pH 6), samples were immunostained with specific antibodies, using the Leica Microsystems’ Bond-max automated immunostainer and the Bond Polymer Refine Detection System (Leica Microsystems), developed with diaminobenzidine and counterstained with haematoxylin (antibodies used, dilutions and optimised incubation times: online supplemental table S4-C). Positive and negative control tissues for protocol optimisation were selected from Human Protein Atlas (www.proteinatlas.org) and obtained from Institut d'Investigacions Biomèdiques August Pi i Sunyer Biobank.

Tissue-microarray (TMA) slides containing 58 HCC samples were purchased from SuperBioChips Laboratories (Seoul, Korea, www.tissue-array.com). Immunohistochemistry (IHC) for OPN and EGFR on serially sectioned TMA slides was carried out using the BOND-MAX automated immunostainer (Leica Microsystems, Bannockburn, IL, USA) with the Bond Polymer Refine detection kit (Leica). Anti-OPN antibody (1:50; R&D Systems) or anti-EGFR antibody (1:100; Ventana Medical Systems, Oro Valley, AZ, USA) were employed as the primary antibody. Each stain was assessed according to the intensity (negative, 0; weak, 1; moderate, 2; intense, 3) and area (none, 0; focal, 1; multifocal, 2; diffuse, 3) of positive cells. The overall grade of each stain was obtained by multiplying the area score by the intensity score (negative, 0; weak, 1 or 2; moderate, 3 or 4; intense, 6 or 9).