After the fixation and permeabilization of cells, samples were processed for the Proximity Ligation Assay (PLA) according to the manufacturer’s instructions (Sigma-Aldrich) using the DuoLink® in situ Orange detection reagent (DUO92102). First, the sample was incubated for 60 min at 37 °C in a humidified chamber in PLA blocking solution. Then the cells were incubated for 1 h at RT with primary antibodies in Antibody Solution: anti-DEK mouse antibody (Santa Cruz, sc-136222, 1:50) and α-H3K9ac (rabbit, Invitrogen, 710293, 1:250), α-H3K4me3 (rabbit, Abcam, Ab8580, 1:250), α-H3K27ac (rabbit, Invitrogen, 720096, 1:500), α-H3K9me2 (mouse, Abcam, Ab1220, 1:200), α-H3K9me3 (rabbit, Abcam, Ab8898, 1:500). After subsequent washing with the Washing Buffer A, we performed the Ligation step for 30 min at 37 °C and Amplification step for 100 min at 37 °C in the dark. After the wash with Washing Buffer B and PBS, the cells were incubated with the secondary antibody anti-mouse Alexa Fluor 488 (ThermoFisher, A11001, 1:250), incubated for 20 min at RT with TO-PRO™-3 Iodide (Invitrogen, 1:2000) when necessary, and washed with PBS. All the samples were mounted in ProLong™ Diamond Antifade Mountant.
Duolink in situ detection reagent orange
Manufactured by Merck Group
Sourced in United States
The Duolink In Situ Detection Reagent Orange is a laboratory equipment product. It is designed for the detection and visualization of protein-protein interactions in cells or tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab36823, by Abcam (2 mentions)
Deltavision system, by Cytiva (2 mentions)
Duolink in situ pla probe anti mouse plus, by Merck Group (2 mentions)
Duolink in situ pla probe anti rabbit minus, by Merck Group (2 mentions)
A11001, by Thermo Fisher Scientific (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab1220, by Abcam (1 mentions)
Sc 136222, by Thermo Fisher Scientific (1 mentions)
Ab8580, by Thermo Fisher Scientific (1 mentions)
Mouse anti p53 sc 126, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 647 affinipure donkey anti mouse igg2b h l, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 affinipure donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Tcs sp5, by Leica (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Sytox green nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Fluoro gel, by Electron Microscopy Sciences (1 mentions)
Bz 9000 microscope, by Keyence (1 mentions)
Duolink in situ pla probe anti mouse minus, by Merck Group (1 mentions)
Duolink in situ pla probe anti rabbit plus, by Merck Group (1 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (1 mentions)
9 protocols using duolink in situ detection reagent orange
1
Proximity Ligation Assay for Epigenetic Markers
2
Quantitative Analysis of DEK-PLA Complexes
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Proximity ligation assay (PLA), including controls, was performed in MCF10A cells as described previously (Pierzynska-Mach et al., 2023 (link)) and using the DuoLink®in situ Orange detection reagent (DUO92102, Sigma-Aldrich) according to the manufacturer's instructions. Cells were incubated for 60 min at 37°C in a humidified chamber in PLA-blocking solution, followed by 1 h at RT with antibody solution containing primary antibodies. After washing, the ligation and amplification steps were performed. Anti-mouse-IgG conjugated to Alexa Fluor 488 was used as secondary antibody. Nascent DNA was detected as described above. Samples were mounted in ProLong™ Diamond Antifade Mountant. Quantitative analysis of ‘PLA-positive’ DEK bodies was carried out by counting cases where the PLA signal was formed in the radius of 1.5 µm from the center of DEK body (Koos et al., 2014 (link)).
3
In Situ Proximity Ligation Assay
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After EdU reaction (see below for details), samples were processed for in situ PLA using the DuoLink in situ Orange detection reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Then, for complete detection of the expression levels of the targeted molecules, samples were incubated with fluorochrome conjugated antibodies. The primary antibodies employed for PLA were: rabbit anti-53BP1 (ab36823, Abcam, Cambridge, UK)/mouse anti-p53 (sc-126, Santa Cruz Biotechnologies, Dallas, TX, USA), and goat anti-p53 (DO1, Santa Cruz Biotechnologies, Dallas, TX, USA)/mouse anti-pH2AX (613402, Biolegend, San Diego, CA, USA). The secondary antibodies were: Alexa488 anti-mouse and Alexa647 anti-rabbit or anti-goat conjugated antibodies (715-545-150 and 711-606-152, Jackson-immunoresearch, West Grove, PA, USA).
4
In situ PLA for Protein Interactions
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Samples were processed for in situ PLA using the DuoLink in-situ Orange detection-reagent (Sigma-Aldrich, Merck Life Science, Milan, Italy) according to the manufacturer’s instructions. The primary antibodies employed in the PLA assay were rabbit polyclonal anti-53BP1 (ab36823, Abcam, Cambridge, UK) and mouse monoclonal IgG2b DynLL1/Pin1 (MAB2294, R&D systems, Minneapolis, MN, USA). Then, after execution of the PLA reactions leading to the creation of the DNA rolling circles that mark the interaction, cells were incubated with fluorochrome-conjugated secondary antibodies: Pacific Orange Goat anti-Rabbit IgG (H + L) (P31584, ThermoFisher Scientific, Waltham, MA, USA) for High Content Widefield/3D-Confocal Microscopy, or Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG (H + L) (711-545-152, Jackson-immunoresearch, West Grove, PA, USA) for High Content Widefield/3D-SIM Microscopy, and, in both cases, Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG2b (H + L) (115-605-207, Jackson-immunoresearch, West Grove, PA, USA). Fluorochrome conjugated secondary antibodies, competing with the residual fraction of oligo-conjugated ones not involved in the PLA circle formation, allowed the detection of the cell expression levels of the targeted molecules in addition to the interaction spots.
5
Proximity Ligation Assay for Endothelial Cells
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Freshly isolated endothelial tubes were placed on Superfrost Plus slides (Thermo Fisher Scientific) and fixed with 2% paraformaldehyde. For immunofluorescence, mesenteric endothelial tubes were incubated with primary and secondary antibodies as described in Table 1 . For the proximity ligation assay (PLA), endothelial tubes were incubated with Duolink blocking buffer for 30 min at 37°C and then incubated overnight with primary antibodies as indicated in Table 1 . Cells were then incubated with anti-rabbit PLUS and anti-mouse MINUS probes (1:5) for 1 h at 37°C. The omission of one or both primary antibodies served as the negative control. Samples were amplified with Duolink In Situ Detection Reagent Orange (excitation/emission: 554/579 nm; Sigma-Aldrich) for 100 min at 37°C. Endothelial nuclei were stained with Sytox Green nucleic acid stain (1:10,000; #S7020; Invitrogen). Endothelial tubes were mounted with FluoroGel (Electron Microscopy Sciences), and the images were acquired sequentially with a 63×/1.3 glycerol objective by confocal microscopy (TCS SP5; Leica) using Argon (488 nm/∼20 mW), HeNe (543 nm/∼1 mW), and HeNe (633 nm/∼10 mW) class IIIb lasers to determine colocalization.
6
Proximity Ligation Assay for Mitochondrial Protein Interactions
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HeLa cells were cultured on glass slides and fixed with 2% PFA for 20 min. The staining of the PLA was performed following the manufacturer’s instructions with Duolink™ In Situ Detection Reagent Orange, Duolink™ In Situ PLA Probe Anti-Rabbit PLUS, Duolink™ In Situ PLA Probe Anti-Mouse MINUS, and Duolink™ In Situ Mounting Medium with DAPI (Sigma-Aldrich/Merck, St. Louis, MO, USA). As primary antibodies, rabbit-anti-human REV3L (Lifespan Biosciences, Seattle, WA, USA, LS-C368491), mouse-anti-human EndoG (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365359), mouse-anti-human DNA POLγ (Santa Cruz Biotechnology, Dallas, TX, USA, sc-3906349), rabbit-anti-human DNA POLγ (Cell Signaling Techonolgy Inc., Danvers, MA, USA, 13609S) mouse-anti-human TLR4 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-293072), and mouse-anti-human mtTFA (Santa Cruz Biotechnology, Dallas, TX, USA, sc-166965) were used. Images were taken with a Keyence BZ-9000 microscope (Keyence, Neu-Isenburg, Germany) and foci were counted manually.
7
Visualizing PKCε/Dynein Interaction
8
Visualizing AhR/SRC Complexes by PLA
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The proximity ligation assay was used to visualize AhR/SRC complexes in SK28 cells. The cells, grown on glass coverslips, were fixed with 4% PFA in 0.1 M phosphate buffer (15735‐60S, Electron Microscopy Sciences) for 15 min at RT and PLA performed using the Duolink® in Situ detection Reagent Orange (DUO92007), Duolink® in Situ PLA® Probe Anti‐Mouse PLUS (DUO92001), and Duolink® in Situ PLA® Probe Anti‐Rabbit MINUS (DUO92005), SIGMA kits according to the manufacturer's protocol. After blocking, the reaction was performed with the primary antibodies: mouse anti‐AhR (C20, 1/100) and rabbit anti‐SRC (1C12, 1/100). Following the ligation and amplification steps, the coverslips were immobilized on microscopic slides using mounting medium containing DAPI. The ligation step was omitted in the control. Imaging analysis was carried out using a delta vision system (Applied Precision). The number of foci was quantified for at least 30 cells.
9
Visualizing AhR/ARNT Complexes in MCF7 Cells
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The proximity ligation assay was applied in order to visualize AhR/ARNT complexes in MCF7 cells. The cells, grown on glass coverslips, were fixed with 4% PFA in 0.1 M phosphate buffer (15735-60S, Electron Microscopy Sciences) for 15 min at RT and PLA was performed using the kit ((DUO92007) Duolink® in Situ Detection Reagent Orange, (DUO92001) Duolink® in Situ PLA® Probe Anti-Mouse PLUS, (DUO92005) Duolink® in Situ PLA® Probe Anti-Rabbit MINUS, SIGMA) according to the manufacturer’s protocol. After blocking, the reaction with primary antibodies, mouse anti-AhR (C20, 1/100) and rabbit anti-ARNT (1C12, 1/100). Following the ligation and amplification steps, the coverslips were immobilized on the microscopic slides with the mounting medium containing DAPI. In control experiment, the ligation step was omitted. Imaging analysis was carried out using a delta vision system (Applied Precision). Number of foci was quantified in at least 30 cells.
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