Cc1 tris edta buffer

IHC staining for ER, PR, HER2, and ASAP1 was performed on 4 μm thick TMA sections using the Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA). The primary antibodies for ER, PR, and HER2 were as follows: ER (SP1) rabbit monoclonal primary antibody (Roche, Basel, Switzerland), PR (1E2) rabbit monoclonal primary antibody (Roche), HER-2/neu (4B5) rabbit monoclonal primary antibody (Roche), and anti-ASAP1/DDEF1 antibody (7B12) (ab125729, Abcam, Cambridge, UK; diluted 1:200). Heat-induced epitope retrieval was performed with CC1 Tris-EDTA buffer (Ventana Medical Systems, Tucson, AZ, USA) at 100 °C for 80 min. Detection of primary antibody was performed with the OptiView DAB IHC Detection Kit (Ventana Medical Systems). IHC staining for Ki-67 was performed on 4 μm thick TMA sections using the Bond-Max automated immunostainer (Leica BioSystems, Newcastle, UK). Monoclonal (MIB-1) Ki-67 antibody (M7240, Dako, Santa Clara, CA, USA; diluted 1:100) was also used for IHC. Heat-induced antigen retrieval was performed with Bond epitope retrieval solution (Leica BioSystems) after deparaffinization. Detection of primary antigen was performed using the Bond Polymer Refine Detection kit (Leica BioSystems) and 3,3’-diaminobenzidine tetrahydrochloride as a chromogen.

Immunohistochemistry (IHC) was performed on the sections using a Benchmark XT Automated Staining System (Ventana Medical Systems, Tucson, AZ, USA). Anti-cortactin (11381-1-AP, 1:400, Proteintech, Rosemont, IL, USA) was used as the primary antibody according to the manufacturer’s instructions. Heat-induced epitope retrieval was performed using CC1 Tris-EDTA buffer (Ventana Medical Systems, Tucson, AZ, USA). The signal was detected using OptiView DAB IHC Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) containing an endogenous peroxidase blocker. Modified Mayer’s hematoxylin (hematoxylin II) was used as a counterstain. Staining, in which all procedures were performed except the primary antibody treatment, served as the negative control.

We first constructed tissue microarrays (TMAs) to detect the molecular target in many specimens at once [23 (link)]. A representative portion of the tumor in each case was selected during the tissue slide review. The cylindrical cores (measuring 3.0 mm in diameter) were acquired from formalin-fixed, paraffin-embedded tissue blocks (donor blocks) corresponding to the tissue slides and were deposited on recipient TMA blocks (Unitma, Gyeonggi-do, Republic of Korea). Each TMA block consisted of 6 × 5 tumor samples.
TMA blocks were cut into 4 μm thick sections and underwent IHC using the Benchmark XT automated staining system (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s protocol. Heat-induced epitope retrieval was performed with CC1 Tris-EDTA buffer (Ventana Medical Systems, Tucson, AZ, USA). The OptiView DAB IHC Detection Kit (Ventana Medical Systems, Tucson, AZ, USA) was used to block endogenous peroxidase and detect antigen–antibody complexes. Counterstaining was performed using modified Mayer’s hematoxylin (Hematoxylin II). Rabbit recombinant monoclonal SSBP2 antibody (diluted 1:200) (ab177944; Abcam, Cambridge, UK) was used as the primary antibody.