Chromogen red

Paraffin sections (10 μm) were dewaxed and stained histochemically with hematoxylin and eosin (HE). For immunohistochemistry, the paraffin sections were dewaxed and subjected to a heat-induced epitope retrieval step except for sections for prior incubation with anti-B220 (clone RA3-6B2, BD Bioscience, 1:400). Primary antibodies against cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:400) and MPO7 (polyclonal rabbit, Dako, code A0398, 1:1000) were used. This was followed by incubation with biotinylated secondary antibodies (Dianova). For detection, alkaline phosphatase-labelled streptavidin and chromogen RED (both Dako) were employed. For detection of macrophages, sections were subjected to protein-induced epitope retrieval employing protease (Sigma) prior to incubation with anti-F4/80 (clone BM8, eBioscience, 1:800). This was followed by incubation with biotinylated rabbit anti-rat (Dako) secondary antibody. Biotin was detected using alkaline phosphatase-labelled streptavidin (Dako). For visualization of alkaline phosphatase, chromogen RED (Dako) was used. Nuclei were counterstained with hematoxylin (Merck). Negative controls were performed by omitting the primary antibody.

The study was approved by the local ethical review committee (Research ethics committee of the Medical Association Westfalen-Lippe and Westphalian Wilhelms University; ethical vote: 2013-156-f-S). We used tissue microarrays of 362 patients. Of these, 37 were diagnosed as being TNBC by missing expression of ER, PR and HER2. Immunohistochemistry of formalin-fixed, paraffin-embedded tissue microarrays was performed using primary antibody (SFRP1, Epitomics, clone# EPR7003) and biotinylated secondary antibodies (DAKO). Detection was performed using Chromogen Red (DAKO) and H&E (Merck). Slides were embedded with Scientific Cytoseal (Thermo Scientific Fisher).
For immunocytochemistry, cells were fixed with phosphate buffered formalin. Cells were blocked with 10% Aurion (DAKO) in PBS for 1 h. Cells were washed and incubated with primary antibody (β-Catenin, Cell Signaling, # 9587) diluted with Dako REALTM Antibody Diluent (overnight at 4°C). Fluorescent visualization was carried out using suitable Alexa Fluor-conjugated secondary antibody (1:600) together with 4′,6-diamidino-2-phenylindole (1:400) in in Dako REALTM Antibody Diluent) for 1 h at RT.