Immunohistochemical Analysis of Whole Feet

Whole feet were examined by IHC as described previously (11 (link)). Sections were stained with rat anti-mouse Ly6G (catalog number NMP-R14; Abcam, Cambridge, MA, USA) (neutrophils), F4/80 (Abcam) (macrophages), or CD3 (Abcam) (T lymphocytes). Detection used NovaRED secondary antibody (ImmPACT NovaRED peroxidase substrate kit [catalog number SK-4805]; Vector Laboratories, Burlingame, CA, USA). Slides were digitally scanned using an Aperio AT Turbo system (Leica Biosystems), images were analyzed using Aperio ImageScope software (Leica), and cell quantitation was undertaken using open-source image analysis software, QuPath v0.2.3 (25 (link)). Three sections per foot were analyzed to produce a mean value for each mouse.

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Liu X., Poo Y.S., Alves J.C., Almeida R.P., Mostafavi H., Tang P.C., Bucala R., Teixeira M.M., Taylor A., Zaid A, & Mahalingam S. (2022). Interleukin-17 Contributes to Chikungunya Virus-Induced Disease. mBio, 13(2), e00289-22.

Publication 2022

F4/80 ImmPACT NovaRED peroxidase substrate kit Aperio ImageScope software

Antibody Cell Foot Macrophages Mouse Neutrophils Peroxidase T lymphocytes Vector

Corresponding Organization : Griffith University

Other organizations : Universidade Federal de Sergipe, Yale University, Universidade Federal de Minas Gerais

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Variable analysis

independent variables

Antibody used for staining (rat anti-mouse Ly6G, F4/80, or CD3)

dependent variables

Number of neutrophils (Ly6G+)
Number of macrophages (F4/80+)
Number of T lymphocytes (CD3+)

control variables

Sections per foot (three sections per foot were analyzed)
Imaging and analysis methods (digitally scanned using Aperio AT Turbo system, analyzed using Aperio ImageScope and QuPath software)

controls

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

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