Goat anti human ephb4 igg

Prior to detection of EphB4, CD31, or LYVE-1 by immunohistochemistry, tumor cryosections were fixed with acetone for 10 min at −20°C. After blocking with 3% v/v H₂O₂ for 10 min (only for EphB4) and with 2% v/v bovine serum albumin and 0.3% w/v skimmed milk for at least 1 h, tumor sections were incubated with primary antibodies (goat anti-human EphB4 IgG, R&D, AF3038, 0.2 µg/mL; rat anti-mouse CD31 IgG, BD, 550274, 0.08 µg/mL; rabbit anti mouse LYVE 1 IgG, ReliaTech Hamburg, Germany, 705 065 003; 1:200, 1 h, room temperature), ExtrAvidin^®-Peroxidase (Sigma Aldrich; 1:50, 30 min, room temperature), and AEC Substrate Kit (BD). Primary antibodies for CD31 and LYVE 1 were visualized by Alexa Fluor 488-conjugated goat anti-rat IgG (Thermo Fisher Scientific, Langenselbold, Germany, A11006; 10 µg/mL) and Alexa 546-conjugated donkey anti rabbit IgG (Thermo Fisher Scientific, A10040, 10 µg/mL, LYVE-1), respectively, for 1 h at room temperature and subsequent fluorescence microscopy using the microscope AxioImager.A1 (Zeiss, Jena, Germany) and software AxioVision (Zeiss). Distribution of fluorescence dye Hoechst 33342 was analyzed without further processing of tumor cryosections by fluorescence microscopy. Subsequent to fluorescence microscopy, tumor cryosections were stained with Hematoxylin and Eosin (H&E).

Preparation of protein extracts from subconfluent cell cultures or resected tumors using RIPA buffer as well as western blot analysis was performed as described elsewhere [58 (link)]. In brief, 80 µg of proteins were separated in a 8.5% v/v sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Fisher Scientific, Schwerte, Germany) using a semi-dry transfer system (Bio-Rad, Munich, Germany). After blocking with 5% w/v skimmed milk for at least 1 h, PVDF membrane was incubated with goat anti-human EphB4 IgG (R&D, AF3038; 0.2 µg/mL) or rabbit anti EphrinB2 IgG (Novus Biologicals, Littleton, CO, USA, NBP1-48551; 3.1 µg/mL) for 2 h at room temperature and afterwards overnight at 4 °C. Membranes were washed three times and incubated with the appropriate horseradish peroxidase coupled secondary antibodies (rabbit anti-goat IgG, Sigma Aldrich, Taufkirchen, Germany; A5420; goat anti rabbit IgG, Sigma Aldrich, A0545) for 1 h at room temperature. Protein bands were detected with Super Signal West Pico, Dura, or Femto Chemiluminescent Substrate (Fisher Scientific) and the MF-ChemiBis 3.2 imaging system (Biostep, Burkhardtsdorf, Germany). To verify equal protein loading, membranes were stripped and reprobed with mouse anti β-actin IgG (Sigma Aldrich, A5316) and horseradish peroxidase coupled rabbit anti-mouse IgG (Sigma Aldrich, A9044).