Inhibitor cm

Four μm-thick sections of formalin-fixed paraffin-embedded (FFPE) explant tissue were stained for the proliferative marker Ki67. Automated immunohistochemistry (IHC) was carried out on a Discovery XT staining module (Roche). Antigen was retrieved by incubating slides in Cell Conditioning Solution (Roche). Endogenous peroxidase activity was reduced by incubating slides in Inhibitor CM (Roche). Slides were incubated in Ki67 primary antibody (Novocastra; 1:100) or Anit-AR primary antibody (clone G122-434, BD) for 1 h at 37°C followed by HRP-conjugated Discovery OmniMaP HRP secondary antibody (Roche) for 30 min. A ChromoMap DAB kit (Roche) was used for detection. Slides were counterstained in hematoxylin and mounted in DPX. Stained slides were imaged using an Aperio CS2 Digital Pathology scanner (Leica). Regions of interest (i.e. epithelial cells) were manually annotated and the percentage of Ki67 positive cells was determined using Aperio Image Analysis software (Leica) in a minimum of four representative 40× magnification regions containing a total of at least 1000 nuclei. Positive control tissue for Ki67 is shown in Supplementary Figure S8 alongside no primary control staining. Serial sections were also stained for p63 (basal cell marker identifying benign regions) and AMACR (cancer cell marker) to define regions of cancer (Supplementary Figure S9).

Cryosections scanned by MALDI-MSI were subsequently analyzed by automated IHC using the Discovery Ultra Automated Slide Preparation System (Roche). Slides were incubated in 4% formaldehyde for 4 min followed by pretreatment in demasking buffer (pH 8.46) at 95 °C for 4 min. Endogenous peroxidase activity was blocked by incubation with inhibitor CM (Roche) for 8 min. The HER2 rabbit monoclonal antibody 134,182 (Abcam) was applied at 1:2000 dilution for 60 min at 37 °C. For visualization of antibody binding the slides were subsequently incubated with UMap anti-rabbit HRP (Roche) for 16 min followed by treatment with DAB staining solution for 8 min. For counter-staining of the nuclei, Hematoxylin II and Bluing Reagent were used for 8 min and 4 min, respectively. Subsequently, the sections were dehydrated in a graded ethanol series and then incubated twice in rotihistol for 3 min each. Finally, the slides were mounted with Entellan® (Sigma Aldrich) and scanned using an AxioScan Z.1.