FFPE Spheroid Immunohistochemistry for ABCG1 Expression

Formalin-fixed paraffin-embedded (FFPE) sections (4 µm) of spheroids were deparaffinised in xylene (2 min) and rehydrated before antigen retrieval in citric acid buffer (10 mM in ddH₂O, pH6, [33 (link)]). Endogenous peroxidases were blocked using 3% hydrogen peroxide for 5 min and endogenous biotin, biotin receptors and avidin-binding sites blocked using an avidin/biotin blocking kit (Invitrogen, Waltham, MA, USA). Sections were incubated for 1 h with ABCG1 primary antibody (10 µg/mL, PA5-13462, Thermo-Fisher Scientific) or rabbit IgG control (10 µg/mL, Dako) at room temperature, followed by incubation with the secondary antibody. Sections were then incubated with streptavidin–peroxidase (Abcam Plc.), followed by DAB substrate (Dako) for 10 min and nuclei counterstained with haematoxylin.