Comprehensive Immunohistochemistry Protocol

Immunohistochemistry was performed using the IHC Staining Kit (Dako, Santa Clara, CA, USA). Tissue sections were deparaffinized in xylene (Thermo), hydrated in phosphate‐buffered saline (Welgene), and blocked with Background Reducing Solution (Dako). The sections were incubated with anti‐CASQ2 (#NBP1‐87304; NOVUS), anti‐aSMA (#BS70000; Bioworld Technology), anti‐FSP1 (#BS7671; Bioworld Technology), anti‐HIF1α (#NB100‐131; NOVUS), anti‐vimentin (#5741; Cell Signaling Technology), anti‐pan‐cytokeratin (#M3515; Dako), and anti‐ki67 (#9027; Cell Signal Technology) at 4 °C overnight, followed by incubation with horseradish peroxidase‐conjugated anti‐secondary antibody (Dako). The signal was developed using diaminobenzidine and hydrogen peroxide, resulting in a brown precipitate. The sections were counterstained with hematoxylin (Dako), dehydrated, and mounted. The DAB area was quantified using IHC Toolbox in ImageJ software (NIH) with 20 random histological fields from five slides of tumor tissues per group [22 (link)].

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Kim J.H., Lee E., Yun J., Ryu H.S., Kim H.K., Ju Y.W., Kim K., Kim J, & Moon H. (2021). Calsequestrin 2 overexpression in breast cancer increases tumorigenesis and metastasis by modulating the tumor microenvironment. Molecular Oncology, 16(2), 466-484.

Publication 2021

IHC Staining Kit xylene phosphate‐buffered saline Background Reducing Solution anti‐HIF1α anti‐vimentin anti‐pan‐cytokeratin anti‐ki67 hematoxylin

Anti antibody Asma Cell Cytokeratin Hematoxylin Hif1α Horseradish peroxidase Hydrogen peroxide Immunohistochemistry Novus Phosphate Saline Tissues Tumor Vimentin Xylene

Corresponding Organization : Seoul National University

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Variable analysis

independent variables

Anti-CASQ2
Anti-aSMA
Anti-FSP1
Anti-HIF1α
Anti-vimentin
Anti-pan-cytokeratin
Anti-ki67

dependent variables

DAB area

control variables

Tissue sections
Blocking with Background Reducing Solution (Dako)
Incubation with horseradish peroxidase-conjugated anti-secondary antibody (Dako)
Counterstaining with hematoxylin (Dako)
Dehydration and mounting of sections

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