Immunohistochemical Analysis of EMT Markers

Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissue sections; 5-μm thick sections were used. The biopsy paraffin-embedded tissue array was prepared. MT1-MMP was detected using rabbit monoclonal primary antibody (EPITOMICS) at a dilution of 1:400. Slides were incubated with the following primary antibodies overnight at 4 °C: mouse anti-E-cadherin (1:100; DAKO System), anti-vimentin (1:800; DAKO System), anti-N-cadherin (1:400; Abcam), anti-snail (1:800; Abcam), and anti-slug (1:400; Abcam). The specificity of the immunostaining reaction was verified by referring to previously confirmed positive- and negative-control tissue sections. Immunohistochemical staining was assessed by scoring, as described previously18 (link). All patients in this study provided informed consents, and the study was approved by the institutional ethics committee at the First Affiliated Hospital of Shihezi University School of Medicine.

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Pang L., Li Q., Li S., He J., Cao W., Lan J., Sun B., Zou H., Wang C., Liu R., Wei C., Wei Y., Qi Y., Hu J., Liang W., Zhang W.J., Wan M, & Li F. (2016). Membrane type 1-matrix metalloproteinase induces epithelial-to-mesenchymal transition in esophageal squamous cell carcinoma: Observations from clinical and in vitro analyses. Scientific Reports, 6, 22179.

Publication 2016

mouse anti-E-cadherin anti-vimentin anti-N-cadherin anti-snail anti-slug

Antibodies Biopsy Cadherin Cadherin 1 Dilution Embedded paraffin Formalin Institutional ethics committee Monoclonal antibody Mouse Paraffin Patients Rabbit Slug Snail Tissue Vimentin

Corresponding Organization : Capital Medical University

Other organizations : Shihezi University, Johns Hopkins Medicine, Johns Hopkins University

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Variable analysis

independent variables

Primary antibody dilution for MT1-MMP detection (1:400)
Primary antibody dilution for E-cadherin (1:100)
Primary antibody dilution for vimentin (1:800)
Primary antibody dilution for N-cadherin (1:400)
Primary antibody dilution for snail (1:800)
Primary antibody dilution for slug (1:400)

dependent variables

Immunohistochemical staining intensity and pattern for MT1-MMP, E-cadherin, vimentin, N-cadherin, snail, and slug

control variables

Formalin-fixed, paraffin-embedded tissue sections
5-μm thick tissue sections
Biopsy paraffin-embedded tissue array
Incubation of primary antibodies overnight at 4 °C

controls

Positive controls: Previously confirmed positive tissue sections
Negative controls: Previously confirmed negative tissue sections

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