Ultramap anti rabbit hrp

Triplex staining for keratin 5, keratin7 and p63 was done by automated multiplex IF using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat-induced antigen retrieval with CC1 (#950-500, Ventana) for 64 min at 97 °C, the tissue samples were incubated firstly with Keratin 5 antibodies for 32 min at 37 °C followed by detection with Ultramap anti-rabbit HRP (#760–4315, Ventana) for 12 min followed by visualization with Red610 for 8 min (#760-245, Ventana). Antibody denaturing was performed using CC2 (#950-123, Ventana) for 20 min at 100 °C. Secondly, Keratin 7 antibodies were incubated for 32 min at 37 °C followed by detection with Ultramap anti-rabbit HRP (#760–4315, Ventana) followed by visualization with FAM (#760-243, Ventana) for 4 min. Antibody denaturing was performed using CC2 (#950-123, Ventana) for 8 min at 100 °C. Thirdly, P63 antibodies were incubated for 32 min at 37 °C followed by detection with Ultramap anti-mouse HRP (#760–4313, Ventana) for 12 min followed by visualization with Cy5 for 12 min (#760-238, Ventana). Slides were incubated in PBS with DAPI for 15 min and covered with anti-fading medium (DAKO, S3023).

To determine the identity of the PD‐L1‐positive cells, triple stains were performed by automated multiplex immunofluorescence using the Ventana Benchmark Discovery (Ventana Medical Systems Inc.). In brief, following deparaffinization and heat‐induced antigen retrieval with CC1 (#950‐500, Ventana) for 64 min at 97°C, the tissue samples were incubated firstly with antibodies against either CD45, gastrin, chromogranin‐A, or somatostatin for 32 min at 37°C followed by detection with either Ultramap anti‐rabbit HRP (#760‐4315, Ventana) or Ultramap anti‐mouse HRP (#760‐4313, Ventana) for 12 min, followed by visualization with Red610 for 8 min (#760‐245, Ventana). Antibody denaturing was performed using CC2 (#950‐123, Ventana) for 20 min at 100°C. Secondly, PDL1 SP263 was incubated for 32 min at 37°C followed by detection with Ultramap anti‐rabbit HRP (#760‐4315, Ventana), followed by visualization with FAM (#760‐243, Ventana) for 4 min. Slides were incubated in PBS with DAPI for 15 min and covered with an anti‐fading medium (DAKO, S3023). Antibody information can be found in Table S1.

Alk and Ros1 IHC was performed on 0.6 mm tissue cylinders as previously described [16 (link),17 (link)]. For Alk IHC, the mouse anti-human ALK monoclonal antibody was applied (clone 5A4, Leica Biosystems). Ros1 IHC was conducted using a rabbit anti-human ROS1 monoclonal antibody (clone D4D6, Cell Signaling Technology). For c-Met IHC a monoclonal rabbit anti-human Met antibody was used (clone SP44, Spring Biosciences). All buffers, including pretreatment CC1 standard incubation buffer, secondary antibody (UltraMap anti-Rabbit HRP) and detection system Discovery ChromoMap DAB, were purchased from Roche Ventana (Tucson, AZ). Immunostainings were performed on the automated immunostainer DiscoveryUltra (Roche Ventana).