Tissue arrayer 1

Tumor specimens were collected during routine surgery prior to the initiation of systemic therapy. The specimens were fixed with 10% formalin following the clinical standard procedures of the Department of Pathology. The specimens were embedded in paraffin and stored at room temperature for preservation. Tissue microarray blocks were constructed by punching three representative cores (1.0 mm) from each tumor using a semiautomatic tissue microarrayer (Tissue Arrayer 1, Beecher Instruments Inc., Silver Springs, MD, USA). Fresh frozen CRC tissue was collected during routine primary tumor surgery. The tumor samples were embedded in optimal cutting temperature (O.C.T) medium and snap frozen on dry ice. The samples were cut into 5 µm cryostat-sections and fixed with acetone.

Formalin-fixed and paraffin-embedded surgical tissue samples came from the archives of the Department of Pathology. The patient tissues were de-identified and analyzed anonymously. Histopathologically representative areas of tumor specimens were defined and marked on haematoxylin- and eosin-stained slides. Three cores from each tumor block were sampled with 0.6-mm punchers and embedded in a new paraffin block by a semi-automatic tissue microarrayer (Tissue Arrayer 1, Beecher Instruments Inc., Silver Spring, MD, USA). Of the six tissue-array blocks prepared, each contained 24–192 tumor samples. Sections of 4 μm each were cut and processed for immunohistochemistry.