Biotin sp conjugated goat anti mouse

Histological and immunofluorescence staining on cryosections was performed as described previously (de Melo et al., 2005 (link)). Primary antibodies used were: mouse anti-BrdU (1:200, Chemicon), mouse anti-BRN3A (1:200, Santa Cruz), goat anti-BRN3B (1:200, Santa Cruz), rabbit anti-caspase 3 (1:500, Cell Signaling Technologies), rabbit anti-DLX2 (1:400, C199 affinity purified), mouse anti-ISLET1 (1:600, DSHB, University of Iowa), rabbit anti-phosphohistone H3 (1:1000, Upstate), rabbit anti-PROX1 (1:500, Chemicon), rabbit anti-PAX6 (1:800, Covance) and mouse anti-syntaxin (1:6000, Sigma). Secondary antibodies and fluorescent tertiary molecules used were: FITC-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-rabbit (1:200), biotin-SP-conjugated goat anti-mouse (1:200) (Jackson ImmunoResearch), streptavidin-conjugated Oregon Green 488 (1:200) and streptavidin-conjugated Texas Red (1:200) (Molecular Probes). Negative controls omitted the primary antibody. TUNEL staining used the In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics). Non-radioactive digoxigenin in situ RNA hybridization was performed as described previously (de Melo et al., 2005 (link)).

Paraffin embedded biopsies from squamous epithelium, Barrett’s metaplasia, HGIN and EAC were cut in 3 μm sections and deparaffinized using a series of different concentrated alcohols and xylol. Endogenous peroxidase was blocked by 3% H₂O₂ for 20 min at 4 °C. The slices were cooked in citrate buffer with a pressure cooker for 10 min. Blocking unspecific bindings was performed in several steps, first with 5% NormalGoatSerum (Jackson Immuno Research, Suffolk, UK) for 20 min at room temperature and second with Reagents from the Streptavidin/Biotin blocking kit (GeneTex, Irvine, CA), as described in the manufacturer’s protocol. Diluted 1:50 in 1% BSA in PBS, the monoclonal primary antibody against WNT3A (AM09053PU-N, OriGene Technologies GmbH, Herford, Germany) was incubated over night at 4 °C. Detecting bound antibody, a biotin-SP conjugated goat anti-mouse (Jackson Immuno Research, Suffolk, UK) and a peroxidase conjugated Streptavidin (Jackson Immuno Research, Suffolk, UK), both diluted 1:1000 in 1% BSA in PBS, were utilized for 1 h at room temperature. Results were visualized with diaminobenzidine and counterstained with hematoxylin. Breast cancer tissue was used as positive control. Evaluation was done using a staining index (1 < 30%, 2 < 60%, 3 < 100% stained) with categorization for analyzing the stroma, the squamous epithelium, and the metaplastic or EAC tissue.