Ventana dp200 scanner

Paraffin blocks were sectioned (5 μm) and placed on poly-L-lysine-coated glass slides. The sections were deparaffinized for 1 h at 60°C. To block the activity of endogenous peroxidase, the sections were incubated in methanol containing 0.3% H2O2 for 30 min. Thereafter, every step was followed by three washes in PBS buffer. To block nonspecific antibody binding, slides were incubated for 1 h in 1% BSA in PBS. Sections were incubated with galectin-9 antibody (ab69630, Abcam, Cambridge, MA USA) diluted at 1:400 overnight at 4°C. Then, the sections were incubated with HRP-conjugated anti-rabbit IgG (ab6721, Abcam) for 1 h at room temperature. Staining was visualised by incubating the slides with DAB substrate (ImmPACT DAB Peroxidase substrate, Vector SK-4105, Burlingame, CA USA) according to the manufacturer’s instructions. To distinguish the nuclei, sections were counterstained in haematoxylin solution (HX87960674 Merck, Darmstadt Germany) for 10 sec. Images were obtained using a VENTANA DP200 scanner (Roche Diagnostics, Tucson, AZ USA). The intensity of galectin-9 expression was determined using Image-Pro Image Analysis Software (Media Cybernetics, Rockville, MD USA). The mean density was calculated for each sample and the expression of galectin-9 was reported in lumens.

Hematoxylin and eosin–stained formalin-fixed paraffine-embedded tissue sections of 142 tumors for which corresponding RNAseq data were available were evaluated independently by two pathologists (M.P. Roudier and M.C. Haffner). All slides were digitized using a Ventana DP200 scanner (Roche) and tumor morphologies were assessed and categorized into adenocarcinoma, adenocarcinoma with cribriform morphology, high-grade carcinoma not otherwise specified (NOS), high-grade neuroendocrine carcinoma, carcinoid like tumor, hybrid tumor with neuroendocrine and adenocarcinoma features, high-grade carcinoma with pleomorphic giant cells and high-grade carcinoma with squamoid features. Morphology calls were integrated and in cases of discrepancies, slides were re-reviewed to achieve a consensus.