Romo1 Immunohistochemical Staining and Scoring

Romo1 protein expression was evaluated by immunohistochemical (IHC) staining. We used a BOND-MAX Immunoautostainer (Leica Biosystems, Newcastle, UK) of the staining after preparation of 4-micron-thick sections of paraffin-embedded tumor tissues. Antigen retrieval was performed by heating the slides at 98°C for 20 min and cooling them for 10 min in Epitope Retrieval Solution 1 and 0.01 M citrate buffer (pH 6.0), respectively. Slides were then washed in distilled water and endogenous peroxidase activity was blocked using a Bond Polymer Refine Detection Kit (Leica Biosystems, Newcastle, UK) for 5 min. After washing, the slides were placed in Tris-buffered saline and incubated for 30 min with Romo1 monoclonal antibody (OriGene Technologies, Rockville, USA) at 1:200 dilution. Subsequently, sections were developed with 3,3’-diaminobenzidine chromogen solution for 7 min, counterstained with hematoxylin, and dehydrated. Positive and negative controls were defined using human colon adenocarcinoma tissues and exclusion of the primary antibody, respectively.

Blinded to the clinical information, a pathologist (J-Y Sung) accessed the Romo1 staining intensities under a light microscope at 200 x. Stained cells were considered as positive when the cytoplasmic staining was identified. Staining intensities of individual cells were graded as 0 (no staining), 1 (weak), 2 (distinct), or 3 (strong), and the percentages of cells with these staining intensities were calculated. Finally, histological scores (H scores) were calculated by multiplying the staining intensities by percentages of cells with each staining intensity (possible range, 0–300).