Immunohistochemical Analysis of Tumor Spheroids

Sections (4µm) of paraffin-embedded multicellular tumor spheroids were made; after deparaffinization and rehydration, these were used for immunohistochemical staining for Ki67 (1:1600, clone D2H10, Cell Signaling) and cleaved caspase 3 (Cleaved caspase 3 (Asp175), 1:800, Cell Signaling). Antigen retrieval was performed by incubating sections in citrate buffer (10 mM, pH 6) for 10 min and cooling down for 2 h. Sections were incubated with primary antibody overnight at 4 °C. The next day, sections were incubated with the BrightVision one step detection system poly-HRP anti-mouse/rabbit (VWRKDPVO110HRP, Immunologic, WellMed B.V., Duiven, The Netherlands) for 30 min at room temperature. Sections were washed with PBS and DAB+ Chromogen (K3468, Dako, Agilent Technologies, Carpinteria, CA, USA) was added to each slide for 10 min. Slides were counterstained with hematoxylin, dehydrated, and mounted. For quantification of the stained sections, the percentage of Ki67 or cleaved caspase 3 positive cells was determined using QuPath Software v.0.2.3 on three different sections on each slide [47 (link)].