Cc1 mild buffer

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).

Specimens from resected (intracranial) brain metastasis tissue (n = 64) and available matched extracranial tumor tissue (n = 44, comprising 9 resected primary lung tumors, 9 lung biopsies and 26 lymph node biopsies) were considered for analysis. Each case was reviewed by a pathologist or neuropathologist. FFPE tissue sections of 2–3 µm thickness were used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). IHC staining for PD-L1 was performed using a Leica Bond immunostainer (Leica Biosystems) according to manufacturer’s protocols. Briefly, tissue sections were deparaffinized, rehydrated, and heat-induced antigen retrieval was performed by incubation in CC1 mild buffer (Ventana Medical Systems) for 30 min at 100 °C. Subsequently, tissue sections were incubated with the primary monoclonal antibody rabbit anti-PD-L1 (Clone E1L3N, Cell Signaling, #13684) at 1:200 for 60 min followed by incubation with HRP-conjugated secondary antibody (Leica Biosystems) for 32 min, DAB incubation and counterstaining of nuclei with hematoxylin. Tonsil served as positive control. PD-L1 expression was quantified by assessing the tumor proportion score (TPS), i.e. assessing the percentage of tumor cells with positive membranous staining relative to all vital tumor cells. Only cases with at least 100 evaluable tumor cells were included [25 (link)].

Formalin-fixed and paraffin-embedded tissue samples of CRC were cut into 4 μm sections. A BenchMark XT immunostainer (Ventana Medical Systems, Tucson, AZ) was used for subsequent immunohistochemical staining. For antigen retrieval, sections were incubated in CC1 mild buffer (Ventana Medical Systems, Tucson, AZ) for 30 min at 100 °C. Sections were stained with anti-E-cadherin antibody (clone 4A2C7, 1:50, Zytomed) for 60 min at room temperature, and visualized using the avidin-biotin complex method and DAB. Cell nuclei were counterstained by incubation with hematoxylin and bluing reagent (Ventana Medical Systems, Tucson, AZ) for 12 min each.
Tissue blocks for immunohistochemistry were available for 12 CTNNB1 mutated CRC and 5 CRC with CTNNB1 wild type. All 17 CRCs were MSI-H.
The stains were evaluated using an Olympus BC50 microscope and Olympus PLN 4X/0.1 Plan Achromat Objectives (Olympus Europe Holding GmbH, Hamburg, Germany). Histological images were acquired with the digital camera moticam 3.0 mp (Motic Instruments Inc., California, USA) and Motic Images Plus software (Motic instruments Inc.).