Macrophage and Neutrophil Quantification

Macrophages were detected with an anti-F4/80 antibody (1:50; BD Bioscience, San Jose, CA) and neutrophils were detected with anti-Ly6G antibody (1:50; BD Bioscience) in addition to their respective isotype controls. Following addition of HRP-conjugated secondary antibodies, slides were counter stained with H&E. F4/80 and Ly6G was quantified by digital images of every 5th field of view for the entire colon using Adobe Photoshop as previously described (26 (link)). NF-κB expression was detected by staining with a fluorescently labeled anti-p100/p52 antibody (1:100, Santa Cruz Biotech, TX) or anti-pNF-κB p65 (1:100, Santa Cruz Biotech). Nuclei were counterstained with DAPI. Relative ratios of p52/DAPI were determined using Image J.

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Mackos A.R., Allen J.M., Kim E., Ladaika C.A., Gharaibeh R.Z., Moore C., Parry N.M., Boyaka P.N, & Bailey M.T. (2019). Mice Deficient in Epithelial or Myeloid Cell Iκκβ Have Distinct Colonic Microbiomes and Increased Resistance to Citrobacter rodentium Infection. Frontiers in Immunology, 10, 2062.

Publication 2019

anti-F4/80 antibody anti-Ly6G antibody anti-pNF-κB p65

Anti antibody Antibodies Colon Dapi Isotype Macrophages Neutrophils Nf κb Nuclei P100

Corresponding Organization : The Ohio State University

Other organizations : University of North Carolina at Charlotte, Massachusetts Institute of Technology

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Variable analysis

independent variables

Anti-F4/80 antibody (1:50; BD Bioscience, San Jose, CA)
Anti-Ly6G antibody (1:50; BD Bioscience)

dependent variables

F4/80 quantification
Ly6G quantification
NF-κB expression (p100/p52 and pNF-κB p65)

control variables

Respective isotype controls for anti-F4/80 and anti-Ly6G antibodies
DAPI nuclear counterstain

positive controls

None specified

negative controls

Respective isotype controls for anti-F4/80 and anti-Ly6G antibodies

Annotations

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