Proximity Ligation Assay for p16 and Occludin in Ileum

After routinely dewaxing and hydration, serial paraffin sections of ileum were blocked with sheep serum and detected with antibodies against p16 (#ab211542, Abcam, Cambridge, MA, United States) and occludin (#sc-133256, Santa Cruz Biotechnology Inc., United States). Then, Duolink Proximity Ligation Assay (PLA) in situ fluorescence (Sigma-Aldrich, United States) was performed following the manufacturer’s instructions with Duolink in situ PLA probe anti-mouse PLUS (#DUO92001), Duolink in situ PLA probe anti-rabbit MINUS (#DUO92005), Duolink in situ detection reagents Red (#DUO92008), and Duolink in situ wash buffer fluorescence (#DUO82049). The PLA signal (λex 594 nm, λem 624 nm; Texas Red) was analyzed (Chen et al., 2020 (link)).

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Zhou J., Hou C., Chen H., Qin Z., Miao Z., Zhao J., Wang Q., Cui M., Xie C., Wang R., Li Q., Zuo G., Miao D, & Jin J. (2021). P16INK4a Deletion Ameliorates Damage of Intestinal Epithelial Barrier and Microbial Dysbiosis in a Stress-Induced Premature Senescence Model of Bmi-1 Deficiency. Frontiers in Cell and Developmental Biology, 9, 671564.

Publication 2021

ab211542 Duolink in situ PLA probe anti-mouse PLUS Duolink in situ PLA probe anti-rabbit MINUS Duolink in situ detection reagents Red

Antibodies Assay Buffer Fluorescence Ileum Ligation Mouse Occludin Paraffin Rabbit Serum Sheep

Corresponding Organization : Nanjing Medical University

Other organizations : Plastic Surgery Hospital, Jiangsu Vocational College of Medicine

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Variable analysis

independent variables

Antibody against p16 (#ab211542, Abcam, Cambridge, MA, United States)
Antibody against occludin (#sc-133256, Santa Cruz Biotechnology Inc., United States)
Duolink Proximity Ligation Assay (PLA) in situ fluorescence (Sigma-Aldrich, United States)

dependent variables

PLA signal (λex 594 nm, λem 624 nm; Texas Red)

control variables

Routine dewaxing and hydration of ileum sections
Blocking with sheep serum

controls

Positive control: Not specified.
Negative control: Not specified.

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