Three to four times ten to the four cells were harvested and cultivated in removable 4-well µ-Slides (Ibidi, Martinsried, Germany) for 24 h at 37 °C. Cells were fixed and permeabilized with 4% formaldehyde for 10 min, followed by 0.1% Triton X-100 for 5 min at room temperature. Blocking was performed with 2 drops of Duolink blocking solution (Duolink In Situ Detection Reagents Red, Sigma) per well for 60 min at 37 °C. Thereafter, slides were incubated with primary antibodies diluted in Duolink antibody diluent at 4 °C overnight on an orbital shaker. After washing four times with wash buffer A, PLA probes were added (70 µL/well) for 1 h at 37 °C in a humid chamber. Ligation and amplification were performed according to the manufacturer’s instructions (Duolink In Situ Detection Reagents Red, Sigma). Slides were washed two times with wash Buffer B at room temperature for 10 min, then overnight at 4 °C. After washing, cells were stained with DAPI 1:1000 in PBS for 4 min, and slides were covered with slide slips. Pictures were taken with an Axiovert 100 microscope and analyzed with Image J.
Duolink in situ detection reagents red
Manufactured by Merck Group
Sourced in United States, Germany
Duolink In Situ Detection Reagents Red is a laboratory product developed by Merck Group. The reagents are designed for in situ protein-protein interaction detection and visualization using a proximity ligation assay technique.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880, by Zeiss (10 mentions)
Duolink in situ pla probe anti mouse minus, by Merck Group (5 mentions)
Duolink in situ pla probe anti rabbit plus, by Merck Group (5 mentions)
Duolink blocking buffer, by Merck Group (3 mentions)
Duolink in situ red kit, by Merck Group (3 mentions)
Duolink in situ pla probe anti mouse plus, by Merck Group (3 mentions)
Duolink in situ pla probe anti rabbit minus, by Merck Group (3 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (3 mentions)
Duolink blocking solution, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Anti rabbit minus, by Merck Group (2 mentions)
Duolink in situ pla probe, by Merck Group (2 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (2 mentions)
Duolink anti mouse minus and anti rabbit plus in situ pla probes, by Merck Group (2 mentions)
Phenylmethanesulfonyl fluoride pmsf, by Beyotime (2 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Bicinchoninic acid bca protein assay kit, by Beyotime (2 mentions)
Duolink in situ probemaker plus, by Merck Group (2 mentions)
Anti mouse igg minus, by Merck Group (2 mentions)
45 protocols using duolink in situ detection reagents red
1
Immunofluorescence Staining Protocol
2
In Situ PLA of BCLAF1-SPOP Interaction
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SK-Hep1 cells were seeded in 24-well chamber slides and incubated in DMEM for 24 h. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100, and blocked with Duolink Blocking buffer (Sigma, USA) for 1 h at 37 °C. In situ PLA was performed using the Duolink in situ Red kit (Sigma-Aldrich, USA). Primary antibodies, including anti-BCLAF1 and anti-SPOP, were incubated overnight at 4 °C. On the following day, the secondary antibody was incubated at 4 °C for 1 h, and the Plus and Mines PLA probes were incubated at 37 °C for 1 h. The Duolink In Situ Detection Reagents Red (Sigma, USA) was used to perform ligation and amplification of the PLA. After multiple washes, cells were mounted in Prolong Gold mounting media with DAPI and imaged using a confocal microscope (LSM880, Zeiss, Japan) with a 63*/1.4NA Oil PSF Objective.
3
Proximity Ligation Assay for p16 and Occludin in Ileum
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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)).
4
Detecting LC3B-ATG5 Binding in GBM Cells
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PLA was performed to detect LC3B-ATG5 binding in GBM cells. U87MG and LN229 cells (3 × 104) expressing EGFP-CD9 were seeded on fibronectin (FN, 10 μg/ml; Sigma-Aldrich, Cat. No. F0895, St. Louis, MO, USA)-coated coverslips in 48-well plates for 1 day. The cells were then fixed in 4% PFA for 15 min at RT. Cells were washed twice with 1 × PBS. The fixed cells were permeabilized using 0.05% saponin (Sigma-Aldrich, Cat. No. S7900) in PBS for 30 min. Samples were stained using primary antibodies for 3 h at RT followed by Duo-Link in situ PLA probes with anti-rabbit MINUS (Sigma-Aldrich, Cat. No. DUO92005), anti-mouse PLUS (Sigma-Aldrich, Cat. No. DUO92001), and Duo-Link in situ detection reagents Red (Sigma-Aldrich, Cat. No. DUO92008). Anti-LC3B antibody (rabbit polyclonal antibody, 1:100; Novus Biologicals, Cat. No. NB100-2220, Centennial, CO, USA, RRID:AB_10003146) and anti-ATG5 antibody (mouse monoclonal antibody, 1:100; Santa Cruz Biotechnology, Cat. No. sc-133158, Dallas, TX, USA, RRID:AB_2243288) were used as primary antibodies. Mounted samples were observed using confocal laser scanning microscopy (CLSM) (LSM700; Carl Zeiss, Jena, DEU, Plan-Apochromat × 63/1.40 Oil DIC M27).
5
Proximity Ligation Assay for DNA-RNA Hybrids
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Proximity ligation assay (PLA) was performed with Duolink In Situ PLA® Probe Anti-Rabbit PLUS (DUO92002, Sigma-Aldrich), Duolink In Situ PLA® Probe Anti-Mouse MINUS (DUO92004, Sigma-Aldrich) and Duolink In Situ Detection Reagents Red (DUO92008, Sigma-Aldrich), according to manufacturer’s instructions. The dissected gonads were freeze-cracked with liquid nitrogen before fixation with cold methanol for 10 min. Anti-DNA–RNA hybrid (mouse, 1:50) and anti-RAD-51 (rabbit, 1:1000) antibodies were used.
6
Activation of TGF-β Signaling in TH17 Cells
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In vitro differentiated sorted TH17 cells were rested for 5 days in Click’s medium supplemented with 2 ng/mL murine IL-2 (PeproTech #212-12) and were subsequently challenged with 1 μg/mL human TGF-β1 for 30 min. Subsequently, the cells were washed, re-suspended in a minimal volume of PBS supplemented with 10% FBS 0.3% BSA, overlaid onto coverslips and were left to dry for 12 h. The cells were rehydrated with PBS, fixed with 4%PFA and permeabilized with 0.3% PBS-Triton X100. After washing, the specimens were blocked, incubated with pospho-Smad3 and Smad4 antibodies and stained with Duolink® In Situ detection Reagents Red (Sigma). After mounting, the slides were analysed by confocal microscopy and the acquired images were opened and processed with Fiji software (Image J).
7
Proximity Ligation Assay for SETD1B and ZEB1
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The PLA was performed according to the manufacturer’s manual and the following reagents were used: Duolink® In Situ Detection Reagents Red (Sigma-Aldrich, Darmstadt, Germany; #DUO92008-100RXN), Duolink® In Situ PLA® Probe Anti-Mouse PLUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92001-100RXN) and Duolink® In Situ PLA® Probe Anti-Rabbit MINUS (Sigma-Aldrich, Darmstadt, Germany; #DUO92005-100RXN). In short, the cells were co-stained over night at 4 °C using the following antibodies: SETD1B (Abcam, Cambridge, UK; #ab113984; 1:1000) and ZEB1 (R&D Systems, Wiesbaden, Germany; #639914; 10 µg/ml). The next day, the PLA probe solution was added to the cells as described in the protocol. After the ligation and amplification steps, the nuclei were stained using ProLong® Gold Antifade reagent (Life Technologies, Darmstadt, Germany). Detection was performed using a Nikon Eclipse Ti-S fluorescence microscope (Nikon, Tokyo, Japan).
8
Proximity Ligation Assay for Protein Interactions
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For visualization of protein–protein interaction in situ, Proximity Ligation Assay (PLA) was performed according to manufacturer's instructions (Duolink using PLA Technology, Sigma). Briefly, 48 h after transient HA-TET1 and/or myc-GADD45a and myc-NFYC (control protein) transfection, HEK293T cells were fixed in 100% MeOH (Sigma) for 10 min at −20 °C, permeabilized with 0.5% TritonX100 (Sigma) for 10 min, blocked and incubated with primary antibodies anti-HA (Abcam) and anti-myc (Sigma) (both diluted 1:1000) overnight at 4 °C. Then, cells were incubated with respective secondary antibodies (Duolink In Situ PLA Probes Anti-Mouse and Anti-Rabbit both PLUS and MINUS, SIGMA) followed by a ligation and subsequent amplification step to visualize protein interaction by red fluorescence signals (Duolink In Situ Detection Reagents Red, Sigma). To visualize individual proteins by Self-PLA, the respective primary antibody was combined with two secondary antibodies (PLA probes both PLUS and MINUS). Nuclei were counterstained by DAPI-containing mounting medium (Duolink In Situ Mounting Medium with DAPI, Sigma) and analysis was performed using a TCS SP5 confocal microscope (Leica) and 63× oil immersion objective lens. The speckles are likely limited to a fraction of cells due to incomplete transfection efficiency.
9
Puro-PLA Procedure for Protein Localization
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The Puro-PLA procedure was performed as previously described (18 (link)). Briefly, after puromycylation with 3 μM puromycin for 15 min, neurons were fixed with 4% paraformaldehyde (PFA)–sucrose for 20 min at room temperature (RT). After fixation, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, blocked in 10% FBS in PBS for 30 min, and incubated with primary antibody pairs diluted in blocking buffer for 1.5 hours at RT. PLA probes (Duolink In Situ PLA Probes, Sigma-Aldrich) were used according to the manufacturer’s instructions. Ligation and amplification reactions were performed according to the manufacturer’s recommendations (Duolink In Situ Detection Reagents Red, Sigma-Aldrich). After amplifications, cells were postfixed in 4% PFA-sucrose for 10 min at RT for better signal stability, processed further for immunocytochemistry with Hoechst staining for nuclei visualization, and mounted with fluorescent mounting medium (Dako).
10
Proximity Ligation Assay for Muscle
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Single myofibres or muscle sections were fixed (4% paraformaldehyde in PBS pH 7.5) and permeabilised (0.1% Triton X-100, 0.1M Glycine, PBS) for 10 min. Permeabilised samples were blocked with the Duolink Blocking Solution (Sigma-Aldrich) for 3 h. Primary antibodies prepared in Duolink Blocking Solution (Sigma-Aldrich) were incubated overnight at 4 °C, then PLA reactions were run using the Duolink PLA probes for mouse and rabbit and Duolink In Situ Detection Reagents Red (Sigma-Aldrich), following the manufacturer’s protocol. After some wash steps, myofibres were mounted on glass slides with Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA).
Quantification of PLA was performed by counting PLA puncta per fibres and whole muscle sections. PLA experiments were performed in TA myofibres isolated from 3 different mice after which a minimum of 15 myofibres or 15 sections were analysed.
Quantification of PLA was performed by counting PLA puncta per fibres and whole muscle sections. PLA experiments were performed in TA myofibres isolated from 3 different mice after which a minimum of 15 myofibres or 15 sections were analysed.
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