Rck105

Immunohistochemistry was performed on a Leica Bond system using the standard protocol F30. The sections were pre-treated using heat mediated antigen retrieval with citrate-based buffer (pH 6, epitope retrieval solution 1) or EDTA based buffer (pH 9, epitope retrieval solution 2) for 20 min. The sections were then incubated with antibody for 30 min at room temperature and detected using an HRP conjugated polymer system in which DAB was used as the chromogen. The sections were counter-stained with haematoxylin and mounted with Aquatex. The following antibodies were used; anti-Pentraxin 3/PTX3 1/500 [MNB1] (Abcam 90806), anti-Cytokeratin 7 1/400 [RCK105] (Abcam 9021), anti-ITGA7 1/500 (Abcam 203254), anti-MGP 1/500 (Abcam 86233), anti-TEM1 1/400 (Abcam 67273), anti-CD31 1/800 [JC70A] (DAKO 20057487), anti-CD68 1/400 (DAKO 20058607), anti-Smooth muscle actin 1/1000 (Abcam 5694), anti-periostin 1/1500 [EPR20806] (Abcam 227049), anti-APOD 1/500 (orb155698), anti-CXCL14 1/1200 (Abcam 137541), anti-Podoplanin 1/750 [D2-40] GTX31231 (lot 821903108).

To detect human breast cancer cells in the mouse organs, immunohistochemical analysis was performed using a human specific cytokeratin 7 antibody (Abcam, clone RCK105, 1:50 dilution) or HLA (Abcam, clone EMR8-5, 1:50 dilution). The paraffin-embedded tissue was deparaffinized in xylene and rehydrated through a series of different graded ethanol, followed by heat induced antigen retrieval in 1 mM EDTA (pH 8.0) or 10 mM sodium citrate buffer (pH 6.0) for 20 min in the 2100 retriever (Aptum) for cytokeratin 7 and HLA, respectively. The slides were allowed to cool to room temperature for 30 min. After the slides were washed, exogenous peroxidase activity was blocked by incubating the slides in 3% peroxidase for 20 min. The slides were then washed, blocked in 5% BSA for 30 min at room temperature, and incubated overnight at 4°C in primary antibody diluted in 5% BSA. Following primary antibody incubation and washing, the slides were incubated in secondary biotinylated anti-mouse IgG reagent (Dako) at room temperature for 1 hour, washed with PBS and then detected using Envision+ (Dako). Counterstaining with hematoxylin was performed and the slides were dehydrated through graded ethanol and then xylene and mounted with Entelen (Electron Microscopy, Inc.).