Envision technique

Immunostaining of esophagus sections for Ki-67, γ-H2AX, 8-OHdG, CD45, CD4, and CD8 was performed on formalin-fixed paraffin-embedded (FFPE) esophagus sections using the Envision technique, Dako Real EnVision Detection System, and Peroxidase/DAB+ (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol. See Method S1 for more details.

Paraffin-embedded sections and slides of 4 mm thickness were deparaffinised and treated with 3% H₂O₂ to block endogenous peroxidase activity. Proteolytic antigen retrieval was performed in pepsin for 30 min at 37 °C, followed by incubation in 10% bovine serum albumin (HyClone, USA) in PBS at room temperature for 10 min to block the non-specific antibody-binding sites. The sections were then incubated overnight at 4 °C with mouse monoclonal antibody against the human RSK4 protein (1:50 dilution; Sigma, USA), human CD44 protein (1:150 dilution; Sigma, USA), human MMP-9 protein (1:300 dilution; Sigma, USA) and humanβ-catenin protein (1:100 dilution; Sigma, USA). Later, a standard rapid EnVision technique (Dako, Denmark) was used to detect the protein conjugates and develop the colour. Finally, the sections were visualised after counterstaining with haematoxylin. Serial sections of RCC were run in parallel with the primary antibody replaced by PBS and rabbit IgG1 (Santa Cruz Biotechnology) as blank and negative controls.

Immunohistochemistry on all samples was performed at the Department of Pathology of UIHC. Sections of the formalin-fixed, paraffin-embedded tissue were deparaffinized in xylene, rehydrated in graded alcohols and rinsed. After antigen retrieval, endogenous peroxidase activity was blocked using 3% H₂O₂. The sections were then incubated with antibodies against Ki-67 (Clone MIB-1, DakoCytomation; Carpinteria, CA, USA), which is specific only for replicative DNA synthesis (proliferation) as well as for the replication and repair markers proliferating cell nuclear antigen(PCNA) and replication protein A (RPA32/RPA2) (Abcam Inc., Cambridge, MA, USA), and TK1 (Clone F12, Novus Biologicals, Inc., Littleton, CO, USA), for 30 to 60 min at room temperature. For the detection of bound antibodies, the EnVision technique (DakoCytomation; Carpinteria, CA, USA) with diaminobenzidine as a substrate was used. The percentage of cells positive for nuclear expression of MIB-1, PCNA, RPA32/RPA2 and TK1 in follicles and diffuse areas were counted in 5 × 400 random fields and an average was calculated
[9 (link)]. To include DNA synthesis for both proliferation and repair, all positive cells were counted independent of staining intensity for both PCNA and TK-1
[9 (link)].