DDX41-DNA interaction was assessed via proximity ligation assay (PLA) using the Duolink In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma) according to manufacturer’s instructions. In brief, THP-1 macrophages were fixed with 100% methanol at −20°C for 20 min and permeabilized with 100% acetone for 10 min. After washing with Wash Buffer A followed by blocking with Duolink Blocking Buffer for 1 h at 37°C in a humidified chamber, cells were incubated with primary antibodies (DDX41, 1:250, NEB; and dsDNA, 1:250, Abcam or ssDNA, 1:250, US Biological) overnight at 4°C. The next day, cells were washed with Wash Buffer A, followed by incubation with appropriate Duolink secondary antibodies (Sigma) for 1 h in humidity at 37°C. After washing with Wash Buffer A at room temperature, ligation and amplification steps of the PLA were performed according to the manufacturer’s protocol. After final washes with Wash Buffer B at room temperature, slides were mounted with Prolong Diamond antifade reagent containing DAPI (Invitrogen). The slides were analyzed and imaged under a confocal microscope; here, red fluorescent dots indicate the close proximity of DNA and DDX41 protein.
Prolong diamond antifade reagent containing dapi
Manufactured by Thermo Fisher Scientific
Prolong Diamond antifade reagent containing DAPI is a laboratory product designed to preserve and protect fluorescent signals in microscopy samples. It contains the dye DAPI, which binds to DNA and emits a blue fluorescent signal. The reagent helps to prevent photobleaching, maintaining the integrity of fluorescent labeling in microscopy applications.
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3 protocols using prolong diamond antifade reagent containing dapi
1
DDX41 and DNA Interaction Analyzed by PLA
2
DDX41 and DNA Interaction Analyzed by PLA
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DDX41-DNA interaction was assessed via proximity ligation assay (PLA) using the Duolink In Situ Red Starter Kit Mouse/Rabbit (DUO92101, Sigma) according to manufacturer’s instructions. In brief, THP-1 macrophages were fixed with 100% methanol at −20°C for 20 min and permeabilized with 100% acetone for 10 min. After washing with Wash Buffer A followed by blocking with Duolink Blocking Buffer for 1 h at 37°C in a humidified chamber, cells were incubated with primary antibodies (DDX41, 1:250, NEB; and dsDNA, 1:250, Abcam or ssDNA, 1:250, US Biological) overnight at 4°C. The next day, cells were washed with Wash Buffer A, followed by incubation with appropriate Duolink secondary antibodies (Sigma) for 1 h in humidity at 37°C. After washing with Wash Buffer A at room temperature, ligation and amplification steps of the PLA were performed according to the manufacturer’s protocol. After final washes with Wash Buffer B at room temperature, slides were mounted with Prolong Diamond antifade reagent containing DAPI (Invitrogen). The slides were analyzed and imaged under a confocal microscope; here, red fluorescent dots indicate the close proximity of DNA and DDX41 protein.
3
Immunocytochemistry in INS-1E Cells
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Following treatment with hIAPP and hαsyn, INS-1E cells were washed once with PBS and fixed with 4% formaldehyde for 15 min at room temperature (RT). The cells were washed three times with PBS and then permebilized with 0.15% Triton X-100 in PBS for 15 min at RT. Cells were blocked for 60 min with 3% BSA in PBS and incubated with primary antibodies in 3% BSA/PBS overnight at 4 °C. The cells were washed three times with PBS and subsequently incubated with Alexa-conjugated secondary antibodies for 1 h at RT. Finally, the cells were rinsed three times in PBS and coverslips were mounted with ProLong Diamond Antifade reagent containing DAPI (Invitrogen, P36966). For information on antibodies used, see Supplemental Table 2 .
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