Tissue sections (5-µm thick) were prepared from formalin-fixed paraffin-embedded (FFPE) CRC and CRC-LM samples. Sections were deparaffinized three times in xylene for 5 min, and hydrated through a methanol gradient (100%, 95%, 70%, and 50%). Unspecific peroxidase activity was blocked by incubation in 3% H2O2/90% methanol for 30 min. After the antigen retrieval step (citrate buffer pH6, 95 °C, 40 min), sections were incubated with Protein Block Serum-Free solution (Protein Block Serum-Free Ready-to-Use, catalog no. X0909, Dako, Glostrup, Denmark) at room temperature (RT) for 30 min, and then with an anti-TGFBI antibody (1:200 dilution; Cell Signaling, Danvers, USA; catalog no. 2719) at 4 °C overnight. Next, samples were washed in PBS, and incubated with Histofine MaxPo-Multi HRP-polymer (Nichirei, Tokyo, Japan; cat. no. 414152F) for 30 min. Sections were washed in PBS three times for 5 min and then stained with 3,3'-diaminobenzidine solution (Agilent-Dako, cat. no. GV800). After counterstaining with hematoxylin (Sigma Aldrich, cat. no. MHS32), they were mounted with Eukitt (Orsatech GmbH, Bobingen, Germany). TGFBI expression was scored in accordance with the previously published methodology 18. Briefly, for each section, staining intensity was evaluated using the following scale: 0 = no staining, 1 = weak, 2 = moderate, and 3 = strong. Then, staining extent (i.e. percentage of the positive area relative to the total section) was quantified as follows: 0 = 0%-25%, 1 = 25%-50%, 2 = 50%-75%, and 3 = 75%-100%. The staining intensity and extent scores were multiplied to yield a composite value, called 'IHC score'. Photographs of representative fields were taken under a Leica DM1000 light microscope (Leica, Wetzlar, Germany) and a Leica MC170 HD camera system. Two independent pathologists scored the samples and the mean scores were reported.

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