Quantifying Tumor Microvessels using IHC

Tumor microvessels were detected by IHC ofFFPE tumors using a polyclonal antibody to CD31 (ab28364; Abcam) at a dilution of 1:500 for 1 hr at RT in primary antibody diluent (Dako). After washing in Tris-buffered saline containing 0.05% Tween-20 (TBS-T), slides were incubated with biotinylated anti-rabbit labeled polymer (Dako). IHC was visualized using DAB peroxidase substrate kit (Dako) and counterstained with hematoxylin (Sigma-Aldrich). The area of tumor microvessels was quantitatively measured using the ImagePro Plus 4.5 software (Media Cybernetics) as previously described [66 (link)]. Tumor section images were imported into the ImagePro Plus software, where the CD31-positive staining was selected using the color selection function. Positive staining pixels were measured using the area/density (intensity) measurement function and represented as microvessel density.

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Blanco F.F., Preet R., Aguado A., Vishwakarma V., Stevens L.E., Vyas A., Padhye S., Xu L., Weir S.J., Anant S., Meisner-Kober N., Brody J.R, & Dixon D.A. (2016). Impact of HuR inhibition by the small molecule MS-444 on colorectal cancer cell tumorigenesis. Oncotarget, 7(45), 74043-74058.

Publication 2016

ab28364 DAB peroxidase substrate kit hematoxylin ImagePro Plus 4.5 software

Antibody Dilution Hematoxylin Microvessels Peroxidase Polymer Rabbit Saline Tumor Tween 20

Corresponding Organization :

Other organizations : Thomas Jefferson University, The University of Kansas Cancer Center, University of Kansas Medical Center, Sidney Kimmel Cancer Center, Savitribai Phule Pune University, University of Kansas, Novartis (Switzerland)

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Variable analysis

independent variables

Dilution of polyclonal antibody to CD31 (ab28364; Abcam) at 1:500

dependent variables

Area of tumor microvessels
Microvessel density

control variables

Incubation time of 1 hr at RT in primary antibody diluent (Dako)
Washing in Tris-buffered saline containing 0.05% Tween-20 (TBS-T)
Incubation with biotinylated anti-rabbit labeled polymer (Dako)
Visualization using DAB peroxidase substrate kit (Dako)
Counterstaining with hematoxylin (Sigma-Aldrich)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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