S3309

Tissue sections from breast cancer specimens were first dried with isopropanol (Fisher Scientific, A461-1) before staining. The sections were then stained with Mayer’s hematoxylin (Agilent, S3309) for 4 min, washed in ultrapure water, incubated in bluing buffer (Agilent, CS702) for 2 min, washed in Milli-Q water and further incubated for 1 min in 1:20 eosin solution (Sigma-Aldrich, HT110216) in Tris-buffer (pH 6). The tissue sections were dried for 5 min at 37 °C and then mounted with 85% glycerol (Merck, 104094) and a coverslip. Imaging was performed using the Metafer VSlide system at ×20 magnification.

The protocols used in our study have previously been described in Ståhl et al.^{24 (link),70 (link)} and a detailed version of the entire protocol is available in Nature Protocols. In short, fresh frozen material was sectioned at 16 μm. After placing the tissue on top of the barcoded microarray, the glass slide was warmed at 37 °C for 1 min for tissue attachment and fixed in ~4% formaldehyde (Sigma-Aldrich, F8775) for 10 min at room temperature (RT). The slide was then washed briefly with 1× PBS (phosphate-buffered saline, Medicago, 09-9400). The tissue was dried with isopropanol (Fisher Scientific, A461-1) before staining. The tissue was stained with Mayer’s hematoxylin (Agilent, S3309) for 4 min, washed in Milli-Q water, incubated in bluing buffer (Agilent, CS702) for 2 min, washed in Milli-Q water, and further incubated for 1 min in 1:20 eosin solution (Sigma-Aldrich, HT110216) in Tris-buffer (pH 6). The tissue sections were dried for 5 min at 37 °C and then mounted with 85% glycerol (Merck, 104094) and a coverslip. Imaging was performed using the Metafer VSlide system at ×20 magnification. The images were processed with the VSlide software (v1.0.0). After the imaging was complete, the coverslip and remaining glycerol were removed by dipping the whole slide in Milli-Q water followed by a brief wash in 80% ethanol and warming for 1 min at 37 °C.