Optiview dab kit

TYRP1 protein expression was centrally assessed at a College of America Pathologist-Clinical Laboratory Improvement Amendments certified laboratory (Ventana Medical Systems, Tucson, AZ, USA) with an investigational TYRP1 immunohistochemistry (IHC) assay, using the Abcam clone EPR13063 as primary antibody (Abcam, Cambridge, UK). Testing was performed on the Ventana Benchmark Ultra platform with the Optiview DAB kit as the detection system (Ventana Medical Systems, Tucson, AZ, USA). Two different cut-offs for TYRP1 tumor cell expression were applied to determine participant eligibility. For the first 12 enrolled participants, at least 25% TYRP1-tumor cell expression at intensities equal to or greater than IHC 1+ was applied. After protocol amendment, based on emerging data from the ongoing trial and additional validation efforts undertaken as part of the IHC development, the cut-off was lowered to at least 1% TYRP1-tumor cell expression at intensities equal to or greater than IHC 1+ for the remaining participants (n = 8). The H score was obtained by the formula: 3 × percentage of IHC 3+ (strongly stained cells) + 2 × percentage of IHC 2+ (moderately stained cells) + 1 × percentage of IHC 1+ (weakly stained cells), giving a range of 0 to 300.

IHC was done on all CRC and EC tissue samples to detect the loss of MMR protein expression as a golden standard test using the following antibodies: MLH1 (clone ES05, diluted 1:50; Dako/Agilent, Santa Clara, CA), MSH2 (clone G219-1129, diluted 1:400; BD Biosciences, San Jose, CA), MSH6 (clone EPR3945, diluted 1:200; Abgent, San Diego, CA), and PMS2 (clone EP51, diluted 1:50; Dako/Agilent, Santa Clara). The MSH2 and MSH6 stainings were performed with Ventana BenchMark ULTRA immunostainer (Roche, Ventana Medical Systems, Tucson, AZ, USA) utilizing OptiView DAB kit (760-700, Ventana/Roche). MLH1 and PMS2 stainings were performed with Autostainer (Agilent/Dako, Santa Clara, USA) utilizing BrightVision detection kit (DPVB110HRP, Immunologic, WellMed, Duiven, the Netherlands). The loss of one or more MMR protein was defined as a dMMR, and the expression of all four MMR proteins was defined as proficient MMR (pMMR). Negative MMR protein expression was considered valid if nuclear staining in the tumor cells was absent with positive external (normal colon mucosa) and internal control staining (stromal nonneoplastic cells).

Immunohistochemistry was performed using an automated single-staining procedure (Benchmark Ultra; Ventana Medical Systems). Briefly, 4 μm thick sections were stained using mouse monoclonal anti-human antibodies directed against CD68 (clone PG-M1, Dako, 1:50) and FR-β (clone OTI8G1, Origen, 1:50). Detection was completed with respective secondary antibodies and the OptiView DAB Kit (Ventana Medical Systems).

Immunohistochemistry of SSTR2 on FFPE lung sections from ILD patients (n = 53) and controls (n = 26) was performed as previously described (Schniering et al, 2019a) using an automated single‐staining procedure (Benchmark Ultra; Ventana Medical Systems) and a rabbit anti‐SSTR2 antibody (abcam, ab134152, 1:50). Detection was finalized with respective secondary antibodies and the OptiView DAB Kit (Ventana Medical Systems). Images were acquired with the AxioScan.Z1 slidescanner (Zeiss) using a Plan‐Apochromat 20×/0.8 M27 objective. For quantification of SSTR2 tissue expression, six randomly selected high power fields were taken per sample with the Zen 2.0 lite (blue edition) software and the percentages of positively stained pixels were automatically quantified using an in‐house designed MATLAB script (MathWorks, MATLAB R2016b). For correlation of SSTR2 expression with the extent of lung fibrosis on the tissue level, the Ashcroft score was applied on adjacent picrosirius red‐stained lung sections as previously described (Schniering et al, 2018, 2019b), and Pearson correlation analysis with SSTR2 tissue expression was performed.

The immunohistochemical (IHC) staining was performed to verify the SSTR2 expressions in the cell lines HEK and HEKsst₂ with and without stimulation of epidrugs. The following combinations of 5-aza-dC/VPA concentrations were used, respectively: 0 µM/1.85 mM, 0.1 µM/1.85 mM, 3.9 µM/1.85 mM and 5.0 µM/1.85 mM. Each sample of HEK and HEKsst₂ cells was transferred into cell blocks. Cell blocks were cut into 1 µm sections, and automatically stained with hematoxylin-eosin staining (Dako Omnis, Aligent Technologies, Santa Clara, CA, USA). SSTR2 IHC staining was performed using the monoclonal antibody anti-SSTR2A (Zytomed Systems GmbH, Bargteheide, Germany) in a dilution of 1:25. The detection was realized by an OptiView DAB Kit (760–700, Roche Holding AG, Basel, Switzerland) using the secondary antibody cocktail, goat-anti-mouse and goat-anti-rabbit, following the manufacturer’s instructions. All above-described immunostainings were processed with the Benchmark ULTRA IHC/ISH System (Roche). Cell sections were covered by an automated cover-slipper (Klinipath, Duiven, Netherlands), digitalized and analyzed by the case viewer software 2.4 (Sysmex GmbH, Norderstedt, Germany). Positive staining of SSTR2 expressions was defined as a brown staining pattern of cell membranes.

IHC for FASN was conducted on 4-µm-thick sections from the JHU and PCBN TMAs utilizing a rabbit monoclonal antibody for FASN (Cell Signaling Technology, catalog no. 3180, RRID: AB_2100796) and the Ventana Benchmark immunostaining system (Ventana/Roche; RRID:SCR_021254). To enable antigen retrieval, slides were incubated with CC1 retrieval solution at 100°C for 32 minutes, and the primary antibody was incubated for 40 minutes at a dilution of 1:100. Detection and counterstain reagents used were the OptiView DAB kit (Roche, 760-700; RRID: AB_2833075), hematoxylin and bluing reagents, respectively. The IHC assay was validated using two positive NCI-60 control cell lines (SK-MEL-5, RRID: CVCL_0527 and T47D, RRID: CVCL_0I95) with high FASN RNA expression based on RNA-seq and another NCI-60 cell line with low FASN RNA expression (RXF-393, RRID: CVCL_1673; ref. 31 (link); Supplementary Fig. S1).
FASN-stained TMA slides were scanned at 20x magnification on the NanoZoomer HT Scanner (RRID:SCR_021658). After scanning, each TMA slide was de-cover-slipped, double stained with p63 [mouse monoclonal (4A4), Abcam, #ab735, RRID: AB_305870,1:100, OptiView DAB kit] to distinguish basal cells identifying benign glands and rescanned.