Gill 2 haematoxylin

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.

Expression of CD56, NKp30 and BAG6 was determined by immunohistochemistry. 3 μm (human) or 5 μm (baboon)-thick paraffin sections were incubated with either monoclonal mouse anti-human CD56 (NCAM, clone 1B6 Novocastra Leica Biosystem, Newcastle, UK) antibody at 1:50, polyclonal goat anti-human NKp30 antibody (sc-20,477, Santa Cruz Biotechnology, Inc) at 1:100 dilution or polyclonal rabbit anti-human BAG6 antibody (HPA053291, ATLAS antibodies, Cambridge Biosciences UK) at 1:500 for 1 h at room temperature in a humidified chamber. Detection was with ImmPRESS anti-mouse, anti-goat or anti-rabbit polymer (Vector Laboratories, Peterborough, UK) respectively and visualisation was with ImmPACT DAB (Vector Laboratories, Peterborough, UK). The sections were counterstained in Gill 2 Haematoxylin, dehydrated, cleared and mounted in Consul Mount (Thermo Scientific, Runcorn, UK). Mouse, goat or rabbit negative control IgG (0.5 μg/ml Vector Laboratories, Peterborough, UK) replaced the respective primary antibody as a negative control.