Goat anti mouse alexafluor 555 secondary antibody

A single 4 μm–thick section was cut from each pretreatment tumor tissue block, deparaffinized in xylene, rinsed in ethanol, and rehydrated. Heat-induced epitope retrieval was performed at 121 °C in citrate-based buffer (Dako) for 6 minutes in a decloaking chamber (Biocare Medical). Slides were stained using an autostainer (Dako). Endogenous peroxidase activity was quenched with peroxidase block for 5 minutes (Dako). Slides were washed with tris-buffered saline Tween-20 wash buffer (Dako) then incubated at room temperature for 30 minutes with signal stain protein block (Cell Signaling) containing a 1:1500 dilution of MRE11 rabbit monoclonal antibody clone EPR3471 (Epitomics) and pan-cytokeratin mouse monoclonal antibody (Dako). MRE11 signal was amplified and visualized using horseradish peroxidase-conjugated goat anti-rabbit antibody with cyanine 5 fluorophore-labeled tyramide signal amplification reagent (Akoya Biosciences). Pan-cytokeratin was visualized using a goat anti-mouse Alexa Fluor 555 secondary antibody (Invitrogen). Stained cells were mounted in mounting media with DAPI (4', 6-diamidine-2'-phenylindole dihydrochloride) (Vector Laboratories, Inc). All work was conducted in a clinical laboratory improvement amendments (CLIA)–grade laboratory by CLIA qualified personnel at the pathology tissue core facility at Moffitt Cancer Center.