Immunohistochemical staining for RXRα and PPARγ was previously described [14 (link),15 (link)]. Briefly, sections were first cut and prepared from paraffin-embedded BC samples using standard protocols, followed by incubation in blocking solution (ZytoChem Plus HRP Polymer System Kit, ZYTOMED Systems GmbH, Berlin, Germany). Hereafter incubation with primary antibodies against RXRα [14 (link)] (PP-K8508-00, Perseus Proteomics Inc., Tokyo, Japan) or PPARγ [15 (link)] (ab59256, Abcam, Cambridge, UK) with a 1:100 dilution for 16 h at 4 °C was performed. Following incubation with a biotinylated secondary anti-rabbit IgG antibody and the associated avidin-biotin-peroxidase-complex (both Vectastain Elite ABC Kit; Vector Laboratories, Burlingame, CA, USA), visualization was performed with 3,3-diamino-benzidine (DAB; Dako, Glostrup, Denmark). Counterstaining of sections with acidic hematoxylin and immediately mounting with Eukitt (Merck, Darmstadt, Germany) was followed by manual analysis with a Diaplan light microscope (Leitz, Wetzlar, Germany) with 25× magnification (see Supplementary Table S1 for precise protocols). A digital CCD camera system (JVC, Tokyo, Japan) was used for obtaining pictures.
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