Protocol detail

Immunofluorescence Analysis of Vascular Morphometrics

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For the morphometric analysis of vasculature, sections from spleens and bone marrow from WT and Ats1-KO mice were subjected to immunofluorescence staining with a monoclonal rat anti-mouse Endomucin antibody (SC-65495, SCBT). Images were captured with the AxioImager A1 microscope (Zeiss), and converted to binary for further analysis with Image J software as indicated^{9 (link)}. For additional immunofluorescence determinations, sections were incubated with the following antibodies: polyclonal rabbit CD31 (AB28364, Abcam), polyclonal goat Nid1 (AF2570, R&D), polyclonal rabbit anti-mouse Versican V1 (2701534, Millipore), polyclonal rabbit anti-mouse Versican DPE (Versikine) (Ab19345, Abcam). Fluorescence confocal images were captured with a LSM 710 confocal microscope (Zeiss).

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Rodríguez-Baena F.J., Redondo-García S., Peris-Torres C., Martino-Echarri E., Fernández-Rodríguez R., Plaza-Calonge M.D., Anderson P, & Rodríguez-Manzaneque J.C. (2018). ADAMTS1 protease is required for a balanced immune cell repertoire and tumour inflammatory response. Scientific Reports, 8, 13103.

Publication 2018

AxioImager A1 microscope AB28364 AF2570 Ab19345 LSM 710 confocal microscope

Anti antibody Antibodies Bone marrow Confocal microscope Endomucin Fluorescence Goat Immunofluorescence Microscope Mouse Nid1 Rabbit Versican

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Variable analysis

independent variables

Genotype (WT vs. Ats1-KO mice)

dependent variables

Morphometric analysis of vasculature in spleen and bone marrow sections
Immunofluorescence staining intensity and localization of Endomucin, CD31, Nid1, Versican V1, and Versican DPE (Versikine)

control variables

Tissue sections (spleen and bone marrow)
Antibodies used for immunofluorescence staining (monoclonal rat anti-mouse Endomucin, polyclonal rabbit CD31, polyclonal goat Nid1, polyclonal rabbit anti-mouse Versican V1, polyclonal rabbit anti-mouse Versican DPE (Versikine))
Microscopy techniques (AxioImager A1 for morphometric analysis, LSM 710 confocal microscope for immunofluorescence)
Image analysis software (ImageJ)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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