RNAscope and Scoring

The RNAscope® 2.5 High Definition (HD)-Red Assay is based on Advanced Cell Diagnostics, Inc. (ACD)’s patented signal amplification and background suppression technology. The assay uses a novel and proprietary method of in situ hybridization (ISH) to visualize single RNA molecules per cell in a multitude of sample types mounted on slides. The RNAscope® 2.5 HD (ACD) was used in accordance with the manufacturer’s instructions [22], with minor modifications (see below). The RNAscope assay was performed on formalin-fixed paraffin-embedded (FFPE) sections using the RNAscope 2.5 HD Assay-RED (322,350, ACD, United States). The paraffin-embedded specimens of ESCC tissues and corresponding paracancerous tissues from each patient were sectioned continuously at 3 μm. The specimens were immediately placed into xylene for dewaxing at 60°C for 1.5 h (2 × 5 min) and subjected to ethanol dehydration (2 × 1 min). RNAscope hydrogen peroxide (322,335, ACD, United States) was added and incubated for 10 min, the slides were placed in boiling (98–102°C) RNAscope target repair reagent (322,000, ACD, United States) for 15 min and immediately rinsed in distilled water (3 times), and hydrophobic circles were drawn when the slides were completely dry (room temperature). RNAscope Protease Plus (322,331, ACD, United States) was added and incubated for 30 min at 40°C in a HybEZ Hybridization furnace (220 VAC, 310,013, ACD, United States). The slides were then hybridized with an F. nucleatum probe (RNAscope® Probe—B-Fusobacterium-23S-3zz, Cat. No. 486411, ACD, United States) for 2 h at 40°C. After hybridization, slides were subjected to signal amplification using the RNAscope® 2.5 HD Detection Kit-RED (322,360, ACD, United States). The tissue sections that had been incubated with the probe were incubated with Amp 1 (preamplifier) for 30 min at 40°C, Amp 2 (background reducer) for 15 min at 40°C, Amp 3 (amplifier) at 40°C, Amp 4 (label probe) for 15 min at 40°C, Amp 5 for 30 min at room temperature, and finally Amp 6 for 15 min at room temperature. After each of these steps, the sections were rinsed in wash buffer (310,091, ACD, United States). Then, the hybridization signal was detected using a mixture of Fast-RED solutions A and B (1:60). After counterstaining with Gill's hematoxylin, slides were dried in a 60°C dry oven for 15 min and sealed with VectaMount (321,584, ACD, United States). The F. nucleatum signal was observed in tumor cells as red granules. As recommended by ACD, a semi-quantitative scoring approach was applied to evaluate the staining results [24]. RNAscope results were examined under a standard bright-field microscope at 20-40× magnification. We used the scoring system provided by the vendor, as follows: 0: negative, 0–1 dots/10 tumor cells at 40×; 1+: 1−3 dots/cell visible at 20−40× magnification; 2+: 4–10 dots/cell visible at 20−40× magnification; 3+: >10 dots/cell and <10% positive cells with dot clusters visible at 20× magnification; and 4+: >10 dots/cell and >10% positive cells with dot clusters visible at 20× magnification. For each slide, five areas containing the highest number of positive cells or dot clusters were selected. All tumor cells within each field were counted, and then the percentage of positive F. nucleatum mRNA signals was used as the final score. Image acquisition and analysis were performed using 3DHISTECH software (automatic digital slide scanner, Pannoramic® MIDI I, 3DHISTECH, HU) and Lucia G software (Laboratory Imaging, Prague, Czech Republic) for microscopic image analysis [25-26].