Tissue Microarray Protocol for Protein Expression

TMA construction by using individual FFPE samples was performed as previously described (8 (link)). Each tissue array block contained up to 50 specimens, which allowed a total of 394 specimens to be mounted on 20 blocks. Serial sections (4 µm) of the FFPE blocks were analyzed for protein expression with immunohistochemistry. A pathologist performed a test procedure by using a control human TMA panel (19 (link)) according to the manufacturer’s information and evaluated the quality of positive and negative controls in immunohistochemistry. Antibodies against RIPK3, MLKL, and PELI1 (F-7) were used due to their essential roles in the necrosome formation and modulation (3 (link)). In addition, antibodies against P53, γH2AX, ATM, Chk2pT68, BRCA1pS1423, and ERCC1 were used due to their well-defined roles in cellular responses to DNA damage (1 (link),8 (link),9 (link)). Information for the antibodies used in this study is summarized in Table S2. All immunohistochemical staining was performed with a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) and the i-View detection kit (Ventana Medical System) by following the standard operating procedure at SuperBioChips Laboratory, Seoul, South Korea.

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Lim J.H., Oh S., Kim L., Suh Y.J., Ha Y.J., Kim J.S., Kim H.J., Park M.H., Kim Y.S., Cho Y., Kwak S.M., Lee H.L., Kim Y.S, & Ryu J.S. (2021). Low-level expression of necroptosis factors indicates a poor prognosis of the squamous cell carcinoma subtype of non-small-cell lung cancer. Translational Lung Cancer Research, 10(3), 1221-1230.

Publication 2021

BenchMark XT autostainer i-View detection kit

Antibodies Cellular Dna damage Human Immunohistochemistry Pathologist Protein Ripk3 Tissue

Corresponding Organization :

Other organizations : Inha University Hospital, Catholic Kwandong University, Inha University, Ajou University

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Variable analysis

independent variables

Antibodies against RIPK3, MLKL, and PELI1 (F-7)
Antibodies against P53, γH2AX, ATM, Chk2pT68, BRCA1pS1423, and ERCC1

dependent variables

Protein expression with immunohistochemistry

control variables

FFPE blocks
Serial sections (4 µm) of the FFPE blocks
Control human TMA panel (19 (link))

positive controls

Control human TMA panel (19 (link))

negative controls

Control human TMA panel (19 (link))

Annotations

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