Bond max autoimmunostainer

Slides were prepared for a representative section of each of the 373 formalin-fixed and paraffin-embedded (FFPE) tissue samples; FFPE tissue section (4-µm-thick) were prepared and stained with H&E using an automatic stainer for routine diagnostic purposes. Representative slides containing deeply invasive tumor parts were selected to visually assess the TSR and tumor pT stage.
For multiplexed H&E- and IHC-stained sections, IHC staining for cytokeratin (CK) (Novocastra™ Liquid Mouse Monoclonal Antibody with 1:500 dilution) was performed. Intermediate filaments found in epithelial cells of all types and markers for carcinoma cells were first analyzed using BOND-MAX Autoimmunostainer (Leica Biosystems, Melbourne, Australia). The IHC-stained slides were scanned at × 200 total magnification (tissue-level pixel size, 0.32 μm/pixel) with Aperio Digital Pathology Slide Scanner (Aperio Technologies, Inc., Vista, CA). After scanning, the same slides were stained with H&E after removing aminoethylcarbazole (AEC) by washing with distilled water, and then, the slides were incubated with 70%, 80%, and 95% ethanol for 2 min each, followed by incubation with 0.15 M KMnO₄ for 1 min as previously described^{20 (link)}. A total of 15 pairs of multiplexed H&E and CK whole-slide images (WSIs) of gastric carcinoma were prepared for this study.

Briefly, 4 μm tissue sections were deparaffinized and rehydrated, and antigens were retrieved for 40 min in a citrate buffer (pH 6.1) at 95°C. We used panTRK (C17F1 Rabbit mAb; 1: 100 dilution; Cell Signaling) as the primary antibody incubating 15 min with Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia). DAB was used as the chromogen, and the sections were counterstained with hematoxylin. For positive control, we used KM12 cell line, which is known to have a TPM3-NTRK1 rearrangement.

For tissue microarray, we reviewed all H&E-stained slides and representative histological areas were carefully selected and marked on paraffin blocks. From each paraffin block, four primary gastric cancer tissue cores (diameter = 0.6 mm) were taken from the invasive front, both lateral sides, and the luminal surface area of the tumor using AccuMax (IsuAbxis, Seoul, Korea) as previously described [22 (link)]. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections using rabbit monoclonal antibody IKKε (D20G4, Cell Signaling Technology, Danvers, MA, USA, 1:50 dilution) and TBK1/NAK (D1B4, Cell Signaling Technology, Danvers, MA, USA, 1:200 dilution). For IKKε, we incubated primary antibody overnight at 4°C and used a DAKO Envision™ Detection Kit (DAKO, Glostrup, Denmark) for 30 minutes. For TBK1, we incubated primary antibody for 15 minutes with Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia) using Bond™ Polymer refine detection (DS9800, Vision Biosystems, Melbourne, Australia) according to the manufacturer’s protocol. For the interpretation of IKKε and TBK1 Immunohistochemistry, strong, distinct cytoplasmic staining with membranous accentuation was considered positive.

The H&E-stained specimens in each case were microscopically reviewed and each case was pathologically diagnosed according to the 2015 WHO classification [5 ]. An appropriate FFPE block containing the tumor in each case was selected by reviewing H&E-stained specimens and each FFPE block was sliced into 3-μm-thick tissue sections. The tissue sections were deparaffinized and subjected to immunostaining with the following four anti-PD-L1 antibodies: clone SP142 (Ventana Medical Systems, Tucson, AZ, USA), clone SP263 (Ventana Medical Systems), clone 22C3 (Dako, Carpinteria, CA, USA), and clone 28-8 (Dako). SP142 and SP263 immunostaining was carried out with a Bond-Max autoimmunostainer (Leica Microsystems, Wetzlar, Germany) and a Bond polymer-refine detection kit (Leica Microsystems). 22C3 and 28-8 immunostaining was carried out with a Dako autostainer Link48 system (Dako) and a PD-L1 PharmDx kit (Dako).

An appropriate formalin-fixed and paraffin-embedded (FFPE) block containing the tumor in each case was selected by reviewing H&E-stained specimens, and each FFPE block was sliced into 3-μm-thick tissue sections. The tissue sections were deparaffinized and subjected to immunostaining with the following for anti-PD-L1 antibodies: clone SP142 (Ventana Medical Systems, Tucson, AZ, USA), clone SP263 (Ventana Medical Systems), clone 28-8 (Dako, Carpentaria, CA, USA), and clone 22C3 (Dako). SP142 and SP263 immunostaining was carried out with a Bond-Max autoimmunostainer (Leica Microsystems, Wetzlar, Germany) and a Bond polymer-refine detection kit (Leica Microsystems). 28-8 and 22C3 immunostaining was carried out with a Dako autostainer Link48 system (Dako) and a PD-L1 PharmDx kit (Dako).