Figure 3

A: Immunohistochemistry (IHC) of alpha-Smooth muscle actin (α-SMA, prediluted mouse monoclonal anti-α SMA, Abcam, Cambridge, UK) in a Stage IIIC colorectal carcinoma with high PRL-3 mRNA expression [8]. Representative slide photography showing intense staining of stromal cells corresponding to CAFs. Briefly, mouse monoclonal anti-α SMA antibody was diluted 1/3 in PBS and incubated for 1 hour at room temperature. After washing steps, reaction was visualized using EnVision anti-mouse antibody system, and developed using DAB-Plus Kit (Dako, Copenhagen, Denmark). Slides were counterstained with Harry's modified hematoxylin. As a negative control, we used EnVision anti-mouse antibody system which displayed no reactivity against any antigen. Positive and negative controls were included in each assay. For IHC quantification, we evaluated non-adjacent tumoral regions to the muscular layer to avoid confusing results. Expression was evaluated according to intensity (1 = -, undetectable or + light; 2 = ++, moderate; 3 = +++, intense staining) and percentage of stain (1 = < 10%; 2 = 10–50%; 3 = > 50%). A score containing the information for both parameters [(intensity +1) × percentage] was assigned, classifying staining as "low" or "high". B: Intense deposition of collagen fibrils in a high mRNA PRL-3 expression Stage IIIC colorectal carcinoma, using Masson trichrome staining (Sigma-Aldrich). To evaluate staining we considered the percentage of tissue occupied by collagen fibrils in relation to total tissue area in each slide without taking into account the muscular layer (< 5% absence; 5–15% slight; 15–50% moderate and > 50% intense deposition). C: Slight deposition of collagen fibrils in a Stage IIIC colorectal carcinoma with previously documented low levels of mRNA PRL-3 expression [8].