Digital slice scanner

Sectioned slides were incubated at 37 °C for 1 min and fixed in methanol for 10 min at −20 °C. For staining, the sections were incubated in isopropanol (MilliporeSigma, Burlington, MA, USA) for 6 min, Mayer’s hematoxylin (Agilent, Santa Clara, CA, USA) for 7 min, Bluing Buffer (Dako) for 1 min, and eosin (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:5 in Tris-base (0.45 M Tris, 0.5 M acetic acid, pH 6.0) for 1 min. The slides were washed with deionized water after each of the staining steps. After air-drying, the slides were mounted with 85% glycerol and coverslips. Hematoxylin and eosin-stained images were recorded at 40× magnification using a digital slice scanner (Hamamatsu, San Jose, CA, USA). The coverslip was removed after imaging by immersing the slides in RNase-and DNase-free water.

The 10 × Visium protocol was optimized for frozen tissue. Briefly, tumours were frozen in dry ice immediately after harvesting. Tumours were embedded with optimal cutting temperature (OCT) compound and cryosectioned at 10-μm thick. The sections on the capture areas were placed and incubated them at 37 °C for 1 min and then fixed them in methanol for 10 min at − 20 °C. To stain, sections were incubated in isopropanol (Millipore Sigma) for 6 min, Mayer’s haematoxylin (Dako, Agilent, Santa Clara, CA) for 7 min, bluing buffer (Dako) for 1 min, and eosin (Sigma-Aldrich) diluted 1:5 in Tris-base (0.45 M Tris, 0.5 M acetic acid, pH 6.0) for 1 min. The slides were washed with deionized water after each of the staining steps. After air-drying, the slides were mounted with 85% glycerol and then coverslipped them. Haematoxylin and eosin (H&E)-stained samples were photographed at 40 × magnification using a digital slice scanner (Hamamatsu). The coverslip was removed after imaging by immersing slides in RNase- and DNase-free water.

Section and staining were performed by the Department of Pathology of the Affiliated Hospital of SWMU. IHC was performed according to standard procedures. The primary antibody, anti-GOLPH2 (also named GOLM1, 1:1000), were used for staining. Results were obtained by digital slice scanner (Hamamatsu Photonics, Hamamatsu, Japan). An immunoreactivity score system was hired to evaluate the expression of GOLM1 (see Supplementary Data A).