Immunofluorescence Microscopy Protocol

Immunofluorescence (IF) was performed as described previously (6 (link), 12 (link), 13 (link)). Briefly, cells were fixed with 4% paraformaldehyde for 30 min and blocked with QuickBlock™ Blocking Buffer (Beyotime, Haimen, China) for 15 min at room temperature. Then cells were incubated with primary antibodies at 4°C overnight, followed by incubation for 2 h at room temperature with AF555 or AF647-conjugated secondary antibody (Biolegend). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using a Nikon AIR Confocal Laser Microscope (Nikon, Minato, Japan) and mean fluorescent intensity (MFI) was measured by a NIS elements AR analysis software version 5.01. All antibodies used in this study are listed in Supplementary Table S2.

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Li R., Zeng X., Yang M., Xu X., Feng J., Bao L., Xue B., Wang X, & Huang Y. (2021). Antidiabetic Agent DPP-4i Facilitates Murine Breast Cancer Metastasis by Oncogenic ROS-NRF2-HO-1 Axis via a Positive NRF2-HO-1 Feedback Loop. Frontiers in Oncology, 11, 679816.

Publication 2021

QuickBlock™ Blocking Buffer Nikon AIR Confocal Laser Microscope

Af647 Antibodies Antibody Ar elements Buffer Cells Confocal microscope Immunofluorescence Nuclei Paraformaldehyde

Corresponding Organization : Chongqing Medical University

Other organizations : Army Medical University, Xinqiao Hospital, University of Colorado Anschutz Medical Campus, The People's Hospital of Guangxi Zhuang Autonomous Region

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Variable analysis

independent variables

Primary antibodies

dependent variables

Mean fluorescent intensity (MFI)

control variables

Cells were fixed with 4% paraformaldehyde for 30 min
Cells were blocked with QuickBlock™ Blocking Buffer (Beyotime, Haimen, China) for 15 min at room temperature
Cells were incubated with primary antibodies at 4°C overnight
Cells were incubated with AF555 or AF647-conjugated secondary antibody (Biolegend) for 2 h at room temperature
Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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