Beecher mta1

Additional hematoxylin and eosin stained sections of normal and tumor were prepared from the selected FFPE blocks and annotated by a GI pathologist (PK) for DNA/RNA extraction and TMA construction. For both DNA and RNA extractions, 6 × 5 micron blank sections were cut from each block and dewaxed in xylene and alcohol. Under direct visualization using magnifying glass annotated areas were scrapped off using a scalpel blade into 1.5 ml tubes. Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega, UK) was used for DNA extraction and RNeasy FFPE Kit (Qiagen, UK) for RNA extraction. Elution was in a volume of 50 ul. TMAs were constructed using 1 mm cores from tumors in triplicate and normals in duplicate on a Beecher MTA1 (Beecher Instruments Inc., WI), following international standards [25 (link)].

Tissue Micro-Arrays (TMAs) were constructed by taking three representative tumour areas from each donor FFPE tissue block on a Beecher MTA1 (Beecher Instruments Inc., WI) as previously described [2 ]. Briefly, representative tumour area selection involves a rigorous selection process by an expert pathologist to identify a tumour area which reflects the characteristics of the tumour as a whole. This process typically requires examination of multiple tissue sections from the donor tissue blocks. Exact areas are annotated and tissue cores obtained in triplicate ensuring that the tumour characteristics are consistent. To control for tumour heterogeneity, section review is conducted consistently to ensure the representativeness of the TMA is retained as it is cut for each study. The manual arrayer was used to extract 1mm cores for insertion into recipient blocks. For IHC 3 micron sections were taken from each TMA slide and stained using previously validated antibodies for CD3, CD4, CD8, CD68, ICOS, IDO1, LAG3 and PD-L1. Staining conditions for all antibodies are listed in Table 1. Automated IHC was performed using a Leica BOND RX (Leica Biosystems, USA) ensuring staining consistency across all samples.