Immunohistochemical Analysis of DPYSL2

Paraffin-embedded tissue microarray was deparaffinized in xylene and ethanol (100%, 90%, and 75%, respectively) and rehydrated. The microarray was boiled in 5,000 mL EDTA (pH = 8.0) for 5 min at high pressure, and endogenous peroxidase was quenched using 0.5% hydrogen peroxide. After washing three times with PBS, the microarray was blocked with 7% normal goat serum and incubated with anti-DPYSL2 antibody (1:100) overnight at 4°C. The results were visualized using a Dako Chem-Mate EnVision kit (K500711; Dako, Agilent Technologies, Santa Clara, CA, United States) and were assessed by two independent pathologists in a blinded manner. The intensity of the staining was scored as previously described (Zou et al., 2019 (link)).

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Zou J., Huang R., Chen Y., Huang X., Li H., Liang P, & Chen S. (2021). Dihydropyrimidinase Like 2 Promotes Bladder Cancer Progression via Pyruvate Kinase M2-Induced Aerobic Glycolysis and Epithelial–Mesenchymal Transition. Frontiers in Cell and Developmental Biology, 9, 641432.

Publication 2021

Dako Chem-Mate EnVision kit

Anti antibody Edta Ethanol Goat Hydrogen peroxide Microarray Paraffin Pathologists Peroxidase Pressure Serum Tissue Xylene

Corresponding Organization : Guangzhou Medical University

Other organizations : Sun Yat-sen University Cancer Center, Sun Yat-sen University

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Variable analysis

independent variables

Deparaffinization in xylene and ethanol (100%, 90%, and 75%, respectively)
Rehydration
Boiling in 5,000 mL EDTA (pH = 8.0) for 5 min at high pressure
Quenching of endogenous peroxidase using 0.5% hydrogen peroxide
Blocking with 7% normal goat serum
Incubation with anti-DPYSL2 antibody (1:100) overnight at 4°C

dependent variables

Intensity of the staining

control variables

Washing three times with PBS

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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