Quantifying Vascular Cell Proliferation and Density

For each tissue section, images were taken at 600x (Ki67 staining) or 200x (SMCA staining) magnification using an Eclipse E600 Nikon microscope and digital camera (Nikon Instruments Inc., Melville, NY, USA) or Zeiss Imager M2 epifluorescence microscope equipped with Zeiss piezo automated stage and AxioCam HRm camera (Carl Zeiss International, Jena, Germany). Image analysis (Image-Pro Plus, Media Cybernetics, Inc., Bethesda, MD, USA) was performed for images of 5–10 randomly chosen fields of CAR to determine vascular cell proliferation based on Ki67 staining, while images of 5–40 randomly chosen fields from areas containing FM or CAR were used to determine the density of blood vessels based on SMCA staining, as described previously (Borowicz et al. 2007 (link), Grazul-Bilska et al. 2010 (link), 2011 (link), 2013 (link)). The LI was calculated as the percentage (%) of proliferating Ki67-positive cells out of the total number of cells within blood vessels which were marked with H and PAS/CAR tissue area.

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Grazul-Bilska A.T., Johnson M.L., Borowicz P.P., Bilski J.J., Cymbaluk T., Norberg S., Redmer D.A, & Reynolds L.P. (2014). Placental development during early pregnancy in sheep: Effects of embryo origin on vascularization. Reproduction (Cambridge, England), 147(5), 639-648.

Publication 2014

Eclipse E600 Nikon microscope Zeiss Imager M2 AxioCam HRm camera Image-Pro Plus

Blood vessels Camera Cell proliferation Cells E600 Microscope Tissue

Corresponding Organization :

Other organizations : North Dakota State University

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Variable analysis

independent variables

Magnification (600x for Ki67 staining, 200x for SMCA staining)

dependent variables

Vascular cell proliferation based on Ki67 staining
Density of blood vessels based on SMCA staining

control variables

Tissue sections
Eclipse E600 Nikon microscope and digital camera
Zeiss Imager M2 epifluorescence microscope equipped with Zeiss piezo automated stage and AxioCam HRm camera
Image analysis using Image-Pro Plus software

controls

No explicit positive or negative controls were mentioned in the input protocol.

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