Histological Analysis of Immune Cells

Formalin fixed, paraffin-embedded histological tissue sections were stained with hematoxylin and eosin (H&E) [19 (link)] or immunohistochemically stained for macrophages (anti-Iba1; Fujifilm Wako catalog # 013–27691) [20 (link)], CD3+ T cells (anti-CD3, Roche, catalog# 790–4341) [21 (link)], and CD4+ T cells (anti-CD4, Abcam ab183685) [22 (link)], as previously described. Sections were also stained for CD8 (Cell Signaling Technology, #98941 clone D4W2Z, Danvers, MA) at 1:300 dilutions, using the UltraVision LP detection system HRP-DAB kit (Thermo Scientific, TL-015-HD). Cytokines in colonic tissue homogenates were measured using Luminex Bead Technology (R&D Systems, Minneapolis, MN) as previously described [23 (link)]. Note that all quantitive histological analyses were done by a reviewer blinded to animal genotype.

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Boucher A.A., Rosenfeldt L., Mureb D., Shafer J., Sharma B.K., Lane A., Crowther R.R., McKell M.C., Whitt J., Alenghat T., Qualls J., Antoniak S., Mackman N., Flick M.J., Steinbrecher K.A, & Palumbo J.S. (2019). Cell type-specific mechanisms coupling protease-activated receptor-1 to infectious colitis pathogenesis. Journal of thrombosis and haemostasis : JTH, 18(1), 91-103.

Publication 2019

anti-CD3 ab183685 UltraVision LP detection system HRP-DAB kit

Animal Anti cd3 Cd4 t cells Cd8 cell Clone Colonic Cytokines Dilutions Eosin Formalin Genotype Macrophages Paraffin T cells Tissue

Corresponding Organization : University of Cincinnati

Other organizations : University of Cincinnati Medical Center, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Animal genotype

dependent variables

Macrophage (Iba1) staining
CD3+ T cell staining
CD4+ T cell staining
CD8+ T cell staining
Cytokine levels in colonic tissue homogenates

control variables

Formalin fixed, paraffin-embedded histological tissue sections
Hematoxylin and eosin (H&E) staining
Immunohistochemical staining protocols

controls

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

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