Immunohistochemical Analysis of Inflammatory Cytokines

Immunohistochemical analysis was performed according to our previous study^{14 (link)}. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min, to remove endogenous peroxidase. Antigen retrieval was performed with the sodium citrate buffer (0.1 M), using the boiling method. The slides were incubated with normal serum to prevent nonspecific binding and then incubated overnight at 4 °C with the following primary antibodies (diluted 1:100 or 1:200): IFN-γ (Santa Cruz, sc-74104), TNF-α (MY BioSource, CA, USA, MBS175453), IL-1β (Santa Cruz, sc-1251), IL-6 (Santa Cruz, sc-7920), IL-10 (Santa Cruz, sc-73309), IL-13 (Santa Cruz, sc-1776), and IL-17 (Abcam, MA, USA, ab79056). The slides were incubated for 2 h with biotinylated secondary antibody (1:500; DAKO, Carpinteria, CA, USA) and horseradish-peroxidase conjugated streptavidin. Signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and cells were counterstained with Mayer’s hematoxylin.

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Lee S.Y., Cho S.S., Li Y., Bae C.S., Park K.M, & Park D.H. (2020). Anti-inflammatory Effect of Curcuma longa and Allium hookeri Co-treatment via NF-κB and COX-2 Pathways. Scientific Reports, 10, 5718.

Publication 2020

IFN-γ TNF-α IL-1β IL-6 IL-10 IL-13 IL-17 biotinylated secondary antibody

Antibodies Antibody Antigen Buffer Cells Chromogen Hematoxylin Horseradish peroxidase Hydrogen peroxide Ifn γ Il 10 Il 13 Il 17 Il 1β Methanol Peroxidase Serum Sodium citrate Streptavidin Tissue Tnf α

Corresponding Organization :

Other organizations : Dongshin University, Mokpo National University, Zhengzhou University, Chonnam National University

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Variable analysis

independent variables

Primary antibodies (IFN-γ, TNF-α, IL-1β, IL-6, IL-10, IL-13, IL-17)

dependent variables

Immunohistochemical signals detected using 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution

control variables

Tissue sections (deparaffinized)
Endogenous peroxidase (removed by treatment with 3% hydrogen peroxide in methanol for 10 min)
Antigen retrieval (performed with sodium citrate buffer, 0.1 M, using the boiling method)
Nonspecific binding (blocked by incubation with normal serum)
Primary antibody incubation (overnight at 4 °C)
Secondary antibody incubation (2 h)
Horseradish-peroxidase conjugated streptavidin
Cell counterstaining (Mayer's hematoxylin)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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