Pannoramic 1000

Formalin-fixed and paraffin-embedded tissue samples of BC were cut into 4 μm sections. For the subsequent immunohistochemical staining, a BenchMark XT immunostainer (Ventana Medical Systems, Tucson, AZ) was used. For antigen retrieval, sections were incubated in CC1 mild buffer (Ventana Medical Systems, Tucson, AZ) for 30 min at 100 °C or in protease 1 for 8 min. The sections were stained with anti-p53 antibody (DO-7, Dako, 1:50), anti-GATA 3 antibody (HG3-31, SantaCruz, 1:50), anti-ER (SP1, Ventana, ready to use), anti-Her2neu (4B5, Ventana, ready to use), anti-CK5/6 (EP24,EP67, abcam, 1:100), anti-CD44 (DF1485, Dako, 1:50), anti-CK20 (KS20.8, Dako, 1:100) and anti-Uroplakin III (AU1, Progen, ready to use) for 60 min at room temperature, and visualized using the avidin–biotin complex method and DAB. A detailed description of the antibodies used for the study can be found in Additional file 1: Table S3. We stained the cell nuclei by additionally incubating for 12 min with hematoxylin and bluing reagent (Ventana Medical Systems, Tucson, AZ).
The stains were evaluated using an Olympus BX50 and Olympus BX46 microscopes (Olympus Europe). Histological images were acquired with the digital slide scanner PANNORAMIC 1000 (3DHISTECH).

Histological assessment of the kidneys was performed on 4-µm-thick paraffin sections that were stained using periodic acid–Schiff (PAS) reagent. Slide digitization was performed using a PANNORAMIC 1000 digital slide scanner (3DHistech, Budapest, Hungary) with a ×20 objective. The whole slide images (WSIs) were analyzed using CaseViewer 2.4 software (3DHistech, Budapest, Hungary). The histology of all glomeruli in a single kidney cross section (minimal 63 glomeruli) was evaluated in a blinded manner. The percentage of affected glomeruli, showing thrombosis and/or hyalinosis within the glomerular capillaries, was scored, and hereby, the percentage of the affected glomerular tuft area was measured.