Rabbit anti human cd31 antibody

Labeled streptavidin-biotin method was used in immunohistochemistry (IHC). PEDF expression, Ki₆₇ antigen and microvessel density (MVD, CD31) were analyzed with human serpin F1/PEDF antibody (R&D, USA), rabbit anti-human Ki₆₇ antibody (AbcamPLC, Boston, MA, USA) and rabbit anti-human CD31 antibody (AbcamPLC, Boston,MA, USA). Briefly, 4–5 μm sections were made from paraffin-embedded tumor tissue specimens of each group and subsequently deparaffinized by sequentially washed with xylene (I and II), 100% ethanol, 95% ethanol, 85% ethanol, 75% ethanol and water. Endogenous peroxide was blocked with 3% H₂O₂ kept in dark place at room temperature for 10 min. Antigen retrieval was done by heated in a pressure cooker in 10 mM sodium citrate buffer (pH 6.0). After washed with PBS, tissues were blocked with goat serum for 30 min at 37 °C then incubated with primary antibody overnight at 4 °C. After washed with PBS for three times, the secondary antibody conjugated to horse radish peroxidase (HRP) was added. HRP was detected with 3,3′-diaminobenzidine substrate (DAB Kit, ZSGB-Bio, Beijing, China) for 30 s or more, terminated by water and re-dyed with hematoxylin (Beyotime Institute of Biotechnology, Shanghai, China) for 30–60 s. 10 random fields at ×400 magnification were examined for each section.

Immunohistochemical staining of CD31 and TdT-mediated dUTP nick end labeling staining was performed as previously described.^{5 (link),31 (link)} For double immunofluorescence examination of CD31 and α-smooth muscle actin (α-SMA), sections were incubated at 4 °C overnight with rabbit anti-human CD31 antibody (Abcam) and fluorescein isothiocyanate–conjugated anti-rabbit IgG antibody (1:100; Invitrogen, Carlsbad, CA) for 1 hour. After blocking, sections were incubated with goat anti-human α-SMA antibody (1:100; Abcam) at 4 °C overnight and Alexa fluor 568-labeled anti-goat IgG antibody (1:100; Invitrogen) for 1 hour. Nuclei were counterstained with TO-PRO-3 iodide (1:1,000, Invitrogen). Imaging was performed in a six-border zone region for each sample (z-series Zeiss LSM-510 Meta Confocal Microscope, Carl Zeiss, Jena, Germany) and analyzed with Zeiss LSM Image Browser software version 3.5 (Carl Zeiss).