Quantifying Endothelial Cell Colony Growth

After PFA fixation, immunostaining with rat anti-mouse CD31 antibody (#553370, BD Biosciences) was performed as previously described [38 (link)]. After the primary antibody, cells were incubated with biotin-conjugated anti-rat IgG antibody (Dako, Santa Clara, CA). Then avidin-biotin complexes were formed employing VECTASTAIN Elite ABC-HRP Kits (#PK-6100, Vector Laboratories, Newark, CA). Finally, EC colonies were visualized with 3,3’-diaminobenzidine (DAB) and nickel chloride (NiCl₂). Images were captured with a Canon EOS kiss X7. Areas and diameters of EC colonies were quantified with Fiji software [39 (link)].

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Shimizu S., Iba T., Naito H., Rahmawati F.N., Konishi H., Jia W., Muramatsu F, & Takakura N. (2023). Aging impairs the ability of vascular endothelial stem cells to generate endothelial cells in mice. Angiogenesis, 26(4), 567-580.

Publication 2023

rat anti-mouse CD31 antibody VECTASTAIN Elite ABC-HRP Kits EOS kiss X7

Anti antibody Anti igg Antibody Avidin Biotin Cells Mouse Nickel chloride Vector

Corresponding Organization :

Other organizations : Osaka University

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Variable analysis

independent variables

Immunostaining with rat anti-mouse CD31 antibody (#553370, BD Biosciences)

dependent variables

Areas and diameters of EC colonies

control variables

PFA fixation
Biotin-conjugated anti-rat IgG antibody (Dako, Santa Clara, CA)
Avidin-biotin complexes formed employing VECTASTAIN Elite ABC-HRP Kits (#PK-6100, Vector Laboratories, Newark, CA)
3,3'-diaminobenzidine (DAB) and nickel chloride (NiCl2) for visualization

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