Duolink in situ detection reagents green

Manufactured by Merck Group

Duolink In Situ Detection Reagents Green is a laboratory equipment product designed for protein-protein interaction analysis. It provides a detection system that amplifies weak signals, enabling the visualization and quantification of protein interactions within cells.

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Duolink In Situ Detection Reagent Green (4 citations) Duolink In Situ Detection Reagents (25 citations) Duolink In Situ reagents (18 citations) Duolink reagent (2 citations)

9 protocols using duolink in situ detection reagents green

Proximity Ligation Assay Using Duolink

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The Proximity Ligation Assay (PLA) was performed using Duolink^® In Situ Red Starter Kit Mouse/Rabbit (Sigma, # DUO92101-1KT) and Duolink^® In Situ Detection Reagents Green (Sigma, # DUO92014) according to the manufacturer’s protocol.

Wang L., Song K., Hao W., Wu Y., Patil G., Hua F., Sun Y., Huang C., Ritchey J., Jones C., Liu L., Guan J.L, & Li S. (2021). FIP200 restricts RNA virus infection by facilitating RIG-I activation. Communications Biology, 4, 921.

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Quantification of Protein Interactions by PLA

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Cells were plated on glass coverslips and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. When indicated, cells were incubated with EdU (5-ethynyl-2′-deoxyuridine). PFA-fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min. EdU was coupled with Alexa Fluor 555 or biotin–TEG azide using click chemistry. Primary antibodies against SMC1 (A300-055A; Bethyl), SMC3 (A300-060A; Bethyl), MCM5 (17967; Abcam), PCNA (P8825; Sigma-Aldrich), and biotin (A150-109A; Bethyl or 200-002-211; Jackson ImmunoResearch) were incubated overnight. Probes from Duolink In Situ PLA Probe Anti-Rabbit PLUS (DUO92002; Sigma-Aldrich) and Duolink In Situ PLA Probe Anti-Mouse MINUS (DUO92004; Sigma-Aldrich) were incubated with coverslip for 60 min at 37°C. For ligation (30 min at 37°C) and amplification (100 min at 37°C), Duolink In Situ Detection Reagents Green (DUO92014; Sigma-Aldrich) was used. The cells were mounted on glass slides with Duolink In Situ Mounting Medium with DAPI (DUO82040; Sigma-Aldrich). Cells were analyzed by fluorescence microscopy, and quantification of PLA signal was performed using CellProfiler software (Carpenter et al, 2006 (link)).

Lebdy R., Patouillard J., Larroque M., Urbach S., Abou Merhi R., Larroque C, & Ribeyre C. (2023). The organizer of chromatin topology RIF1 ensures cellular resilience to DNA replication stress. Life Science Alliance, 6(4), e202101186.

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In Situ Protein Interaction Analysis

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PLA was performed using the Duolink^® In Situ Red Starter Kit (mouse/rabbit, DUO92101, Sigma), and Duolink In Situ Detection Reagents Green (DUO92014, Sigma) as described previously [76 (link)]. Briefly, fixed cells or brain sections were permeabilized and blocked with PLA blocking buffer for 1 h at 37 °C and incubated with the indicated primary antibodies overnight at 4 °C. The samples were then incubated with the PLA probes (Anti-Rabbit PLUS and Anti-Mouse MINUS) for 1 h at 37 °C, followed by the ligation and amplification steps. The PLA signal was visible as a distinct fluorescent spot and analyzed by confocal microscopy (Fluoview FV1000, Olympus). The number of fluorescent signals was quantitated using NIH ImageJ software.

Shang Y., Sun X., Chen X., Wang Q., Wang E.J., Miller E., Xu R., Pieper A.A, & Qi X. (2022). A CHCHD6–APP axis connects amyloid and mitochondrial pathology in Alzheimer’s disease. Acta Neuropathologica, 144(5), 911-938.

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Proximity Ligation Assay for Rab7-RILP Interaction

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Duolink Proximity Ligation Assay³⁸ was used to study interaction between Rab7 and RILP. Briefly the primary neurons from WT and Cln1^−/− mice were grown in chamber slides and fixed with methanol for 15 to 20 min at −20°C. The cells were blocked with goat serum and incubated with Rab7 antibody (Abcam; Cat#ab50533; dilution 1:1000) and RILP antibody (Abcam; Cat#ab140188; dilution 1: 3000) overnight at 4°C. After three washes with PBS the cells were incubated with anti-rabbit-MINUS (Sigma–Aldrich; Cat#DUO92005 Anti-RILP) and anti-mouse–PLUS (Sigma–Aldrich; Cat#DUO92001-for Rab7 antibody) PLA probes and subjected to ligation and amplification reaction using Duolink® In Situ Detection Reagents Green (Sigma–Aldrich; DUO9201) according to manufacturer’s protocol. For controls, cells were similarly processed but without primary antibody or with one primary antibody only. When the primary antibodies were in close proximity (<40 nm apart) fluorescent signals were detected as green dots under the FITC channel. The cells were mounted with DAPI-Fluoromount G (Thermo Fisher, Cat#010020) and visualized using an LSM710 confocal microscope. High resolution z stack images were captured at 40X, the z stacks were merged by maximum intensity projection using Zen Desk software to include all the PLA signals and quantitated using Duolink image tool.

Sarkar C., Sadhukhan T., Bagh M.B., Appu A.P., Chandra G., Mondal A., Saha A, & Mukherjee A.B. (2020). Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of INCL. Journal of inherited metabolic disease, 43(5), 1082-1101.

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In Situ Proximity Ligation Assay

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Cells were pre-extracted for 5 min on ice and fixed in 2% formaldehyde in PBS (w/v) for 20 min on room temperature. In situ PLA was performed using Duolink PLA technology (Sigma–Aldrich) according to the manufacturer's instructions. Briefly, coverslips were blocked for 30 min at 37 °C and incubated with the respective primary antibodies for 1 h at room temperature. Upon washing the coverslips twice in PBS for 5 min, anti-Mouse PLUS and anti-Rabbit MINUS PLA probes (Sigma–Aldrich) were coupled to the primary antibodies for 1 h at 37 °C. After three wash steps in buffer A (0.01 M Tris, 0.15 M NaCl, and 0.05% Tween-20) for 5 min, PLA probes were ligated for 30 min at 37 °C. Coverslips were then washed three times 5 min in buffer A. Amplification using the “Duolink In Situ Detection Reagents Green” (Sigma–Aldrich) was performed at 37 °C for 100 min. After amplification, coverslips were washed twice in buffer B (0.2 M Tris and 0.1 M NaCl) for 10 min and once in 0.01× buffer B for 1 min. Finally, coverslips were mounted using Vectashield Mounting Media (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole, sealed, and imaged on an Olympus IX81 FL microscope.

Ren J., Wu Y., Wang Y., Zhao Y., Li Y., Hao S., Lin L., Zhang S., Xu X, & Wang H. (2021). CtIP suppresses primary microRNA maturation and promotes metastasis of colon cancer cells in a xenograft mouse model. The Journal of Biological Chemistry, 296, 100707.

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Proximity Ligation Assay for αV and LAP-TGF-β

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The green PLA signal was observed for α_V and LAP-TGF-β interaction. PLA was performed on fixed and permeabilized tumour cells according to the manufacturers’ instructions (DuoLink In Situ Detection Reagents Green, Sigma Aldrich). After blocking, anti-α_V (clone EPR16800, Abcam ab179475, 1/100) and anti-LAP-TGF-β (clone TW7-16B4, BioLegend 141402, 1/100) antibodies were used. PLA-minus and PLA-plus probes, containing the secondary antibodies conjugated with oligonucleotides (dilution in Antibody diluent 1/5), were added and incubated for 1 h at 37 °C. After hybridization, oligonucleotide ligation and a rolling circular amplification were performed. Cell membranes were stained with WGA red conjugate (Thermo Fisher Scientific W21405, 1/1000), nuclei with Fluoromount-G mounting media containing DAPI (eBioscience), and then analyzed with a confocal microscope SP8 (Leica). The number of PLA signals, marked as green dots on the cell surface, was counted in 25−30 fields containing 500−600 cells, by image analysis (Icy, http://icy.bioimageanalysis.org/download/, v2).

Malenica I., Adam J., Corgnac S., Mezquita L., Auclin E., Damei I., Grynszpan L., Gros G., de Montpréville V., Planchard D., Théret N., Besse B, & Mami-Chouaib F. (2021). Integrin-αV-mediated activation of TGF-β regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade. Nature Communications, 12, 5209.

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Quantifying TFAM-VHL Interaction in Cells

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786-0 cells (pRC3 and VHL WT) were plated on glass coverslips. Cells were incubated with 100 nM MitoTracker Red CMXRos (Invitrogen) for 1 h at 37 °C and fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton X-100 (Sigma-Aldrich) in PBS. After incubation with TFAM and VHL antibodies (1:500 dilution; 8076, Cell Signalling Technology; and 1:1,000 dilution; 564183, Becton Dickinson, respectively) at 4 °C overnight, the PLA assay was performed using Duolink In Situ PLA Probe Anti-Rabbit PLUS and Anti-Mouse MINUS, and Duolink In Situ Detection Reagents Green (Sigma-Aldrich) following the manufacturer’s instructions. The immunofluorescence signals were acquired by an LSM 700 Laser Scanning Confocal System with Zeiss Observer Z1 Inverted Phase Contrast Fluorescence Microscope (ZEISS) using ×63 magnification. Twenty images of randomly selected areas per cell line were taken. Each fluorophore channel was pseudo-coloured in ZEN2 (ZEISS), exported as JPEG, and analysed using the CellProfiler 4.2.0 cell image analysis software (Broad Institute of Harvard and MIT). The number of PLA signals per cell was quantified from the maximal intensity projection of each image. Statistical analysis was performed using Prism 9 software to calculate the non-parametric Mann–Whitney U test (GraphPad). A P value < 0.05 was considered significant.

Li S., Li W., Yuan J., Bullova P., Wu J., Zhang X., Liu Y., Plescher M., Rodriguez J., Bedoya-Reina O.C., Jannig P.R., Valente-Silva P., Yu M., Henriksson M.A., Zubarev R.A., Smed-Sörensen A., Suzuki C.K., Ruas J.L., Holmberg J., Larsson C., Christofer Juhlin C., von Kriegsheim A., Cao Y, & Schlisio S. (2022). Impaired oxygen-sensitive regulation of mitochondrial biogenesis within the von Hippel–Lindau syndrome. Nature Metabolism, 4(6), 739-758.

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ATM-KAP1 Proximity Ligation Assay

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Proximity ligation assay between ATM and KAP1 protein in sodium butyrate-treated HH514-16 cells was conducted according to manufacturer’s instructions with goat anti-ATM and rabbit anti-KAP1 antibodies (A300-136A and A300-274A, Bethyl Laboratories), Duolink In Situ PLA Probe Anti-Goat MINUS plus Anti-Rabbit PLUS (DUO92006 and DUO92002, Sigma-Aldrich), Duolink In Situ Detection Reagents Green (DUO92014, Sigma-Aldrich) and washing buffers (DUO82049, Sigma-Aldrich).

Li X., Burton E.M, & Bhaduri-McIntosh S. (2017). Chloroquine triggers Epstein-Barr virus replication through phosphorylation of KAP1/TRIM28 in Burkitt lymphoma cells. PLoS Pathogens, 13(3), e1006249.

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Tau-Tubulin Interaction Assay Using Duolink

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Duolink proximity ligation assay for detection of interaction between tau and β-tubulin was conducted using rabbit anti-human tau ab-3 antibodies, mouse anti-human β-tubulin antibodies, Duolink^® In Situ PLA^® Probe Anti-Rabbit PLUS, Duolink^® In Situ PLA^® Probe Anti-Mouse MINUS, and Duolink^® In Situ Detection Reagents Green as suggested by the manufacturer (Sigma-Aldrich). Tau Ab-3 and β1-tubulin antibodies were used at 1:100 dilution.

, & Barbolina M.V. (2022). Targeting Microtubule-Associated Protein Tau in Chemotherapy-Resistant Models of High-Grade Serous Ovarian Carcinoma. Cancers, 14(18), 4535.

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