Envision flex substrate buffer

Serial sections were cut from the selected FFPE tumor blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, DAKO, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min.
Staining was performed using a DAKO Autostainer Link 48 (DAKO).
Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibodies were mouse monoclonal anti-CD3 (code M7254, DAKO) diluted 1:600, and anti-CD8 (code M7103, DAKO) diluted 1:300. The primary antibodies were diluted with Envision Flex antibody diluent (code S2022 DAKO).
Primary antibodies were incubated for 30 min at room temperature, and for amplification Envision Flex + Mouse(Linker) (DAKO) was used for 20 min. Bound antibodies were detected using Envision FLEX/HRP (DAKO) and visualized by Envision FLEX DAB (DAKO) and chromogene diluted in Envision Flex Substrate Buffer (DAKO). To enhance the immunohistochemical stains, the sections were incubated in 0.5% CuSO₄ in TBS buffer pH 7.6 for 10 min. Meyer’s hematoxylin (Merck, Damstadt, Germany) was used as counterstain, and finally, the histological slides were coverslipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Serial sections were cut from the selected tumour blocks (N = 129) and mounted on FLEX IHC Microscope Slides (K8020, Agilent DAKO products, Glostrup, Denmark). The pretreatment processes were performed using PT Link (DAKO). Heat-induced epitope retrieval was achieved with Envision Target Retrieval Solution (DAKO) at pH 9 and 97 °C for 20 min. Staining was performed using a DAKO Autostainer Link 48 (DAKO). Endogenous peroxidase activity was blocked by Envision FLEX Peroxidase-Blocking Reagent (DAKO). The primary antibody was mouse monoclonal cytokeratin AE1/AE3 (code M3515, DAKO) diluted 1:250 with Envision Flex antibody diluent (code S2022 DAKO). Primary antibody was incubated for 30 min. at room temperature, and for amplification Envision Flex+ Mouse (Linker) (DAKO) was used for 20 min. Bound antibodies were detected by Envision FLEX/HRP (DAKO) and visualised by Envision FLEX DAB (DAKO) with chromogen diluted in Envision Flex Substrate Buffer (DAKO). Meyer’s hematoxylin (Merck, Darmstadt, Germany) was used as counterstain and finally, the histological slides were cover slipped with Tissue-Tek PERTEX (Histolab Products AB, Göteborg, Sweden).

Antigen expression of Survivin and Mucin-1 was assessed on archived FFPE blocks of tumor biopsies taken from metastatic lesions. Mucin-1 staining was performed according to a protocol described previously (17 (link)). For Survivin, the procedure was similar. First, 4-μm thickness sections were cut, and slides were deparaffinized and rehydrated prior to antigen retrieval by boiling in EnVision™ FLEX target retrieval solution (pH 9, K8004, Dako) for 10 minutes. Subsequently, endogenous peroxidase was blocked using 3% hydrogen peroxidase (76,051,800.1000, EMD Millipore) in PBS (4391.9010, Klinipath) for 10 minutes. Incubation was performed with a primary anti-Survivin antibody (D8, sc-17779, Santa Cruz Biotechnology, TX, USA, dilution) for 1 hour at room temperature. EnVision™ FLEX Wash Buffer (DM831, Dako) was used to wash between steps. Afterwards, slides were incubated with the secondary antibody BrightVision poly-HRP-anti-Ms/Rb/Rt IgG (DPVO999HRP, ImmunoLogic) at room temperature for 30 min. Finally, incubation with EnVision™ FLEX DAB Buffered Substrate and EnVision™ FLEX Substrate Buffer (K5207 and SM803; DAKO) was done for 10 min at room temperature prior to dehydration, counterstaining with hematoxylin and finally enclosing with Quick-D mounting medium (7281, Klinipath). Finally, a pathologist evaluated antigen expression as either positive or negative.

Tissues used for immunohistochemical staining were fixed in formalin and paraffin-embedded (FFPE). FFPE slides were incubated with monoclonal antibodies against RIG-I or MEX3A (1:100, overnight, at 4 °C). The day after, the slides were incubated for 20 min, with secondary antibodies coupled with peroxidase (Dako). Bound peroxidase was detected by diaminobenzidine (DAB) solution (ScyTek Laboratories, Logan, UT, USA) and EnVision FLEX Substrate buffer containing peroxide (Dako). Cell quantification was performed on collected sections, using the imaging software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V., Florence, Italy). Images were captured by HistoFAXS software (TissueGnostics GmbH, Vienna, Austria), at 20× magnification.