Sections were dewaxed and rehydrated as previously described [9 (link)], followed by heat-mediated antigenic retrieval using a pH 6.0 citrate buffer (BRD2) or a pH 8.0 EDTA buffer (BRD4). Endogenous peroxidase activity was quenched by incubation of the slides in 3% hydrogen peroxide for 30 min; cell membranes were permeabilised by 0.1% saponin in PBS, and non-specific labelling blocked by 5% normal horse serum (BRD2) or 5% normal goat serum (BRD4) in PBS for 20 min, at room temperature (as for all the following steps). After washing in PBS, sections were incubated for 1 h with mouse anti-human BRD2 (sc-393720; Santa Cruz Biotechnology) at a 1∶2900 dilution (0.069 µg/ml) or with rabbit anti-human BRD4 (ab128874; Abcam) at a 1∶300 dilution (1.577 µg/ml). For negative control slides, normal mouse or normal rabbit non-specific immunoglobulins (Santa Cruz Biotechnology) were used at the same protein concentration as the primary antibodies. After repeated PBS washing, sections were incubated with anti-mouse or anti-rabbit biotinylated antibody (Vector ABC Kit, Vector Laboratories) for 30 min. Sections were then incubated with ABC reagent (Vector ABC Kit, Vector Laboratories) for 30 min and stained with chromogen-fast diaminobenzidine (DAB) for 1–5 min. Finally, the slides were counterstained in haematoxylin and mounted on permanent mounting medium.
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