Bluing buffer

Sectioned slides were incubated at 37°C for 1 min., fixed in 3.7%–3.8% formaldehyde (Sigma-Aldrich) in PBS (Medicago) for 10 min, and then washed in 1x PBS (Medicago).
For staining, sections were incubated in Mayer’s hematoxylin (Dako, Agilent, Santa Clara, CA) for 4 min, bluing buffer (Dako) for 30 s, and Eosin (Sigma-Aldrich) diluted 1:5 in Tris-base (0.45M Tris, 0.5M acetic acid, pH 6.0) for 30 s. The slides were washed in RNase and DNase free water after each of the staining steps.
After air-drying, the slides were mounted with 85% glycerol (Merck Millipore, Burlington, MA) and coverslips (Menzel-Gläser). Bright-field (BF) images were taken at 20x magnification using Metafer Slide Scanning platform (MetaSystems). Raw images were stitched with VSlide software (MetaSystems). The coverslip and glycerol were removed after imaging by immersing 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.