Immunohistochemical Staining of Steroid Receptors

After antigen retrieval at pH6 as previously described (Hapangama et al, 2012 (link)) 3 μm formalin-fixed paraffin-embedded tissue sections were immunostained with anti-human steroid receptor antibodies and Ki67; antibody sources, concentrations and incubation conditions are detailed in Supplementary Table 1. Detection was with the ImmPRESS polymer-based system and visualisation was with ImmPACT DAB (Vector Laboratories, Peterborough, UK) used in accordance with the manufacturer's instructions. Sections were lightly counterstained in Gill 2 Haematoxylin (Thermo Scientific, Runcorn, UK), dehydrated, cleared and mounted in synthetic resin. Matching isotype (0.5 μg ml) replaced the primary antibody as a negative control, with internal positive controls performed in each staining run.