IHC was performed on 3 μm-thick sections using standard protocols and an automated IHC staining system (Dako Autostainer Plus; Dako Denmark A/S, Glostrup, Denmark) as described previously.7 (link) The primary antibodies used were those targeting human DAB2IP (clone ab87811, diluted 1:400, rabbit IgG1; Abcam, Cambridge, UK) and p-STAT3 (sc-8059, diluted 1:500, mouse IgG1, Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antigen retrieval was achieved using EDTA buffer (pH 9.0) in a pressure cooker for 4 min for DAB2IP or 20 min for p-STAT3. The stained tissue sections were reviewed and scored separately by two pathologists blinded to the clinical parameters. Discordant cases were discussed using a double-headed microscope to obtain a consensus classification.
The total immunostaining score for DAB2IP and p-STAT3 was calculated as the sum of the percentage of stained cells and the staining intensity, according to previously published reports.7 (link),11 (link) The percentage of stained cells was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (>75%). The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The staining of DAB2IP and p-STAT3 was determined as negative (low expression, a final staining score of <3) or positive (high expression, a final staining score of ≥3).
The total immunostaining score for DAB2IP and p-STAT3 was calculated as the sum of the percentage of stained cells and the staining intensity, according to previously published reports.7 (link),11 (link) The percentage of stained cells was scored as 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (>75%). The staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The staining of DAB2IP and p-STAT3 was determined as negative (low expression, a final staining score of <3) or positive (high expression, a final staining score of ≥3).
