Medical background sniper

Tissue microarrays were purchased from SuperBioChip Laboratories (Seoul, South Korea), and were comprised of 40 matched normal breast cores, 70 breast cancer cores, and 10 matched lymph node cores. The tissues were stained for BAALC expression with a rabbit monoclonal antibody (1:50; Novus Bio, Noble Park North, VIC, Australia), using a Ventana Discovery Ultra automated system (Ventana Medical Systems Inc., Tucson, AZ, USA) as previously described (30 (link)), with the following modifications. Antigen retrieval was performed for 40 min, and Biocare Medical Background Sniper was applied for 32 mins at 35°C. Sections were scored using an immunohistochemistry score on a continuous scale of 0-300, as previously described (31 (link)). Briefly, this involves integrating the intensity and frequency of staining. Staining intensity was scored in four categories: no staining (0), weak staining (1, light brown membrane staining that is visible only under high magnification), intermediate staining (2), and strong staining (3, dark brown staining that is visible under low magnification). The percentage of cells at different staining intensities was determined using HALO software (Indica Labs, Corrales, NM). An H-score was then calculated using:

For tyramide signal amplification (TSA) IHC staining, slides were first deparaffinized and rehydrated in serial passage through xylene and alcohol. Antigen retrieval was performed by microwaving the samples for 2 min 20 s with 100% power, followed by 20% power for 15 min and the slide was cooled for 20 min. Then, the sections were incubated with blocking solution, Biocare Medical Background Sniper supplemented with 2% BSA, for 15 min at room temperature. Slides were incubated with primary antibodies: Ki67 (1:50, Dako pH6), CD20 (1:10,000, EDTA pH9) and Cytokeratin-5 (1:10, Diva), VEGF (1:100, EDTA pH9) for 1 h at room temperature. Multiplexed TSA was visualized using performing a triplex (CD20 in Opal 650, CK-5 in Opal 570,Ki67 in Opal 690, and/or VEGF in Opal 570), followed by a 5-plex (addition of HMGB1 Opal 520). All multiplex TSA analyses were performed by repeating staining cycles in series, microwaving in between each cycle and at the end of the multiplex TSA. Slides were then counterstained with DAPI for 5 min and mounted with VECTASHIELD.