Signal stain protein block

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.

After tissue microarray construction, 4 m thick sections were cut from the TMA block and deparaffinized in xylene, rinsed in ethanol, and rehydrated. Heat-induced epitope retrieval was performed by heating slides to 121°C in a citrate-based buffer (pH 6.0) target retrieval solution (Dako, Mississauga, ON, Canada) for 6 minutes, in a decloaking chamber (Biocare Medical, Concord, CA, USA). Slides were stained using a Dako Autostainer. Endogenous peroxidase activity was quenched with a 10 minute incubation of peroxidase block (Dako) followed by a 15 minute protein block (Signal Stain, Cell Signaling, Danvers, MA, USA) to eliminate non-specific antibody binding. Slides were washed with TBST wash buffer (Dako) and then incubated at room temperature for 60 minutes with Signal Stain protein block (Cell Signaling) containing a 1:500 dilution of mouse anti-ING1 mAb, clone CAb5 (SACRI Antibody Facility, University of Calgary, Calgary, AB, Canada). Additional antibodies including anti-pan-cytokeratin guinea pig monoclonal (Acris, San Diego, CA, USA), anti-vimentin rabbit mAb, clone EPR3776 (Epitomics, Burlingame, CA, USA) and Alexa-488 conjugated goat anti-guinea pig antibody (Invitrogen, Burlington, ON, Canada) were used as suggested by suppliers.

TMA sections (4 μm) were deparaffinized and rehydrated as previously described [43 (link)]. Heat-induced epitope retrieval was performed using a decloaking chamber (Biocare Medical, Concord, CA, USA) by heating slides to 121°C for 1 minute, in a citrate-based (pH 6.0) target retrieval solution (S1699, DAKO, Mississauga, Canada). Immunostaining was performed on a DAKO Autostainer as previously described [44 (link)]. Generation of the Cav3.1 rabbit polyclonal antibody was described previously [45 (link)]. The antibody was diluted to 1:10,000 in Signal Stain^® protein block (8112L, Cell Signaling, Danvers, MA, USA) and applied at room temperature for 60 minutes along with pan-cytokeratin to identify tumor epithelia (mouse clone AE1/AE3, M3515, 1:100, DAKO). Following three washes in TBST, anti-rabbit EnVision+ (K4011, DAKO) secondary antibody containing anti-mouse Alexa 555-conjugated secondary antibody (A21424, 1:200, Thermo Scientific, Burlington, ON, Canada) was applied for 60 minutes, and visualized with TSA-Plus Cy5 signal amplification reagent for 5 minutes (Perkin Elmer, Waltham, MA, USA). After immunostaining, slides were coverslipped using ProLong Gold anti-fade mounting medium with diamidino-2-phenylindole (DAPI) (P36935, Thermo Scientific), and stored at 4°C until scanned.