Dako envision detection kit

Immunohistochemistry (IHC) was performed using the Dako Envision Detection kit (DAKO REAL EnVision) [53 (link)]. Sections were counter-stained with haematoxylin (Leica Microsystems). As negative controls, adjacent or similar sections were processed with antibody diluent (Dako). The antibodies (Abs) used to stain the original patient samples with appropriate control for each Ab are summarised in Additional file 2: Table S2.
A total of 27 Nottingham MB patients diagnosed between January 1986 and January 2006 were analysed by IHC. Consent for tumour tissue use was taken in accordance with national tumour banking procedures (UK: 05/MRE/04/70). A second TMA comprising 233 patient samples was obtained from the DKFZ [48 (link)]. Both TMAs were stained with the anti-ABCB1 Ab (C219, 1:40; Calbiochem) and patients with clear evidence of cell membrane staining were scored as positive by three independent reviewers who were blinded to clinical and patient molecular variables.

Tumor samples were fixed in 4% formalin overnight and prepared as paraffin-embedded stocks. Hematoxylin and eosin staining and immunohistochemistry were performed according to standard procedures. The slides were incubated with primary antibodies against Ki67 (1:200, Abcam), Bcl-2 (1:100, Abcam), Bax (1:100, Abcam), and cleaved caspase-3 (1:100, Abcam) followed by treatment with HRP-conjugated secondary antibody (Dako, Glostrup, Denmark). Immunoreactivity was visualized with the Dako EnVision™ Detection kit (Dako). The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed following the manufacturer’s instructions (Roche, Basel, Switzerland). Briefly, the slides were digested with proteinase K, blocked with H₂O₂, and incubated with the terminal deoxynucleotidyl transferase mixture for 60 min. The slides were thoroughly washed, incubated with streptavidin-HRP, and visualized by diaminobenzidine.

Immunohistochemical staining of HLA class I was performed on TMAs using HLA-ABC (Clone EMR8-5; 1:200 dilution; Abcam) with Dako EnVision detection kit (Dako, Carpinteria, CA, USA). Some of the IBC cases had been evaluated for HLA-ABC expression in the previous study^{36 (link)}, and those cases were re-evaluated during the study. HLA-ABC expression was evaluated in the tumor cell membrane for both intensity and proportion of positive cells. We adopted the interpretation method of HLA class I expression from the study by Na et al.^{19 (link)}. The staining intensity was classified into negative, weak positive, and strong positive. Lymphocytes and endothelial cells were used as internal control, and weak positivity was defined as staining weaker than internal control. Staining equivalent or stronger than internal control was considered strong positive. For statistical analysis, high expression was defined as 50% or more tumor cells showing strong positivity. Low expression was defined as < 50% of the tumor cells revealing strong positivity or ≥ 50% showing weak positivity.

FFPE tissues were cut into 4.5-µm-thick sections using a microtome and immuno-stained with the monoclonal 5G4 anti-mouse antibody [44 (link)], which labels only the disease-associated αSyn (1:4000; 5 min pre-treatment with 80% formic acid; Roboscreen, Leipzig, Germany). Target retrieval was performed using the DAKO EnVision FLEX Target Retrieval Solution. The DAKO EnVision detection kit, EnVision FLEX peroxidase-blocking solution, 3,3′-Diaminobenzidine chromogen, and EnVision FLEX+ mouse linker (Dako, Glostrup, Denmark) was used to visualize the antibody reactions.
Digital images were obtained with TissueScope^TM LE120 and TissueSnap^TM (Huron, Saint Jacobs, ON, Canada) and cropped using HuronViewer software (Figure S1).

Immunohistochemical staining was carried out using the DAKO Envision detection kit (Dako, Carpinteria, CA, USA). In brief, paraffin-embedded tissue blocks were sectioned (4 μm-thick), dried, deparaffinized, and rehydrated. Antigen retrieval was performed in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). For all samples, endogenous peroxidase activity was blocked with a 3% H2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min and incubated with an appropriately diluted primary antibody mouse monoclonal antibody D2-40 (diluted 1:50), anti-PDGF-BB rabbit polyclonal antibody (diluted 1:200) or anti-VEGF-C rabbit polyclonal antibody (diluted 1:100) overnight at 4°C. The slides were then probed with an HRP-labeled polymer conjugated to an appropriate secondary antibody for 30 min. Each step was followed by washing with PBS. Each batch of staining was accompanied by positive and negative control slides. Primary human NSCLC tissues, which are demonstrated to exhibit high levels of PDGF-BB and VEGF-C protein, were used as positive controls. Normal mouse IgG substituted for primary antibody was a negative control.

For tissue microarray, we reviewed all H&E-stained slides and representative histological areas were carefully selected and marked on paraffin blocks. From each paraffin block, four primary gastric cancer tissue cores (diameter = 0.6 mm) were taken from the invasive front, both lateral sides, and the luminal surface area of the tumor using AccuMax (IsuAbxis, Seoul, Korea) as previously described [22 (link)]. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections using rabbit monoclonal antibody IKKε (D20G4, Cell Signaling Technology, Danvers, MA, USA, 1:50 dilution) and TBK1/NAK (D1B4, Cell Signaling Technology, Danvers, MA, USA, 1:200 dilution). For IKKε, we incubated primary antibody overnight at 4°C and used a DAKO Envision™ Detection Kit (DAKO, Glostrup, Denmark) for 30 minutes. For TBK1, we incubated primary antibody for 15 minutes with Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia) using Bond™ Polymer refine detection (DS9800, Vision Biosystems, Melbourne, Australia) according to the manufacturer’s protocol. For the interpretation of IKKε and TBK1 Immunohistochemistry, strong, distinct cytoplasmic staining with membranous accentuation was considered positive.