Ventana automated immunostainer

The placenta and umbilical cord samples were submitted for histological examination in neutral-buffered 10% formalin. They were dehydrated according to the standard techniques, embedded in paraffin, cut to 5-mm, and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed with the labeled streptavidin–biotin peroxidase detection system using the Ventana automated immunostainer (Ventana Medical systems, Tucson, AZ). The following antibodies were tested: CD31 (dilution 1:50), CD34 (dilution 1:50), cytokeratins (AE1/AE3 clone; dilution 1:50); all from Dako, Glostrup, Denmark. Endothelial cells of the umbilical vessels and amnion epithelium of the umbilical cord were used as internal controls for pan-cytokeratin and CD31 antibody respectively.

Patients with NSCLC, adenocarcinoma histology, whose tumors were found to be positive for EML4-ALK fusion gene using IHC, were considered for this study. Permission was obtained from the Ethics Committee before the start of the study. Clinical characteristics and treatment details were collected from the patient's medical records. ALK gene rearrangement was detected by IHC using a Ventana automated immunostainer (Ventana Medical Systems, Illkirch Graffenstaden, France). IHC was assayed on 4 μm neutral buffered formalin fixed; paraffin-embedded tumor tissues using a primary rabbit monoclonal ALK antibody (mAb) clone D5F3 obtained from Ventana USA. IHC staining was performed using a Ventana benchmark XT immunostainer. The slides were dried at 60°C for 1 h, deparaffinized using EZ Prep at 75°C for 4 min, and incubated with the primary mAb at a dilution of 1:50 for 1 h at 37°C. Detection was performed using a multimer technology system with the UltraView Universal DAB detection kit.
The primary endpoint of this study was PR. The width of the resultant confidence intervals (CIs) for parameters to be estimated was constructed with a significance level of 0.05, i.e., a 95% CI. OS and PFS were analyzed with the use of Kaplan–Meier survival analysis and estimates were provided with 95% CIs. Statistical analysis was performed using SAS 8.02 (SAS Institute Inc.).

Immunohistochemistry (IHC) was carried out in the Anatomic and Molecular Pathology (AMP) Core Lab, Department of Pathology & Immunology, Washington University School of Medicine in St. Louis with IRB approval. IHC was performed by a standard protocol on Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Antigen retrieval for anti-Mannose phosphate isomerase antibody (rabbit monoclonal, clone EPR10234, AbCam) was performed with CC2 buffer (Cell Conditioning 2; citrate-based buffer pH 6.0, Ventana Medical System) for 68 min at 95 degrees. Sections incubated with primary antibodies for 40 min at 37 degrees. Biotin-free multimer technology system, based on direct linkers between peroxidase and secondary antibodies (ultraView Universal DAB Detection Kit, Ventana Medical System) was used as detection kit. The PMI staining was validated by Z.Z. and Z.A.

All pathologic slides of lymph node were reviewed by an experienced pathologist to confirm pN0. Formalin-fixed, paraffin-embedded tissue was sectioned at a thickness of 4 µm. The tissues were then stained with CK clone AE1/AE3 (1:200 dilution), Epithelial antigen clone BerEp4 (1:100 dilution), and p53 clone DO-7 (1:5,000 dilution) using a Ventana automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s stated protocol. The quantification of immunoreactivity was assessed using ImageJ Fiji software, version1.2. The immunohistological slides were meticulously reviewed by an experience pathologist with expertise in the field. The pathologist maintained a blinded approach throughout the review process, ensuring they were unaware of any relevant clinical information about the patients.