Anti cd31 primary antibody

Tissue samples were fixed with 4% paraformaldehyde solution, embedded in paraffin, and sectioned. Sections were deparaffinized, microwaved to optimize antigen retrieval, and blocked with 1% fetal bovine serum and 3% peroxide. Sections were incubated with anti-CD31 primary antibody (1:75, R&D Systems, Minneapolis, Minnesota, USA) overnight at 4°C and a secondary antibody (1:200, KPL, Gaithersburg, Maryland, USA) for 1 h at room temperature. The sections were colored with 3,3′-diaminobenzidine tetrahydrochloride (DAB; DAKO, Carpinteria, California, USA) and counterstained with hematoxylin. The continuous positive CD31 signals represented microvessels in tumor tissues. MVD was determined by counting the number of positive microvessel structures under a microscope in five random fields at 400× magnification.

Blood vessels evaluation was carried out as follows: tissue underwent antigen retrieval using 1 mM EDTA supplemented with 0.05% Tween 20, pH 8.0 for 40 min at 95-100° C. After blocking with 5% normal horse serum for 1 hour at RT, sections were incubated with anti-CD31 primary antibody (R&D Systems 1:40) overnight at 4° C, followed by donkey anti-goat secondary antibody (ABCAM 1:200) for one hour at RT. Sections were then mounted with Fluorosheild mounting medium with DAPI (ab104139, ABCAM). Control samples were exposed only to the secondary antibody to rule out non-specific staining.
Stained slides were imaged for fluorescence using the Lionheart™ FX Automated Fluorescent Microscope. Three random fields at X20 magnification were captured for each section. The blood vessels were counted and the mean fluorescence intensity (MFI) was measured. Furthermore, total surface area covered by all the blood vessels was calculated by averaging the two diameters of each vessel and multiplying it by the total number of blood vessels in the same ROI.