Freshly isolated VSMCs were studied using the Duolink in situ PLA detection kit 563 (Olink, Uppsala, Sweden). Cells were adhered to coverslips, fixed in PBS containing 4% paraformaldehyde for 15 min, and permeabilized in PBS containing 0.1% Triton X-100 for 15 min. Cells were blocked for 1 h at 37°C in blocking solution and incubated overnight at 4°C with anti-TRPC1, anti-Gαq, and anti-PLCβ1 antibodies (all at 1:200) in antibody diluent solution. Cells were labeled with combinations of either anti-goat Plus/anti-rabbit Minus or anti-goat PLUS/anti-mouse Minus depending on animal species used for 1 h at 37°C. Hybridized oligonucleotides were ligated for 30 min at 37°C prior to amplification for 100 min at 37°C. Red fluorescently labeled oligonucleotides were then hybridized to rolling circle amplification products and visualized using a confocal LSM 510.
Duolink in situ pla detection kit 563
Manufactured by Olink
Sourced in Sweden
The Duolink in situ PLA detection kit 563 is a laboratory equipment product designed to detect protein-protein interactions within cells. It utilizes a proximity ligation assay (PLA) technique to identify and visualize the interactions between target proteins.
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2 protocols using duolink in situ pla detection kit 563
1
Detecting TRPC1, Gαq, and PLCβ1 Interactions
2
Colocalization of Kv7.4, Caveolin-1, and Dynein
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Colocalization of dynein with Kv7.4 or caveolin-1 and caveolin-1 with Kv7.4 was studied with PLA in HEK293B cells stably expressing Kv7.4 and Kv7.4-Q580A or freshly isolated rat mesenteric artery myocytes using the Duolink in situ (PLA) detection kit 563 (Olink) per the manufacturer’s instructions. Similar to previous studies (Zhong et al., 2010a (link); Brueggemann et al., 2014 (link); Chadha et al., 2014 (link); Jepps et al., 2015 (link); Stott et al., 2016 (link); Barrese et al., 2020 (link)), cells were allowed to adhere to coverslips and fixed in 4% paraformaldehyde in PBS. Cells were permeabilized in PBST, blocked in Duolink blocking solution, and incubated with pairs of primary antibodies. The primary antibodies employed were dynein (ab23905; Abcam), Kv7.4 (ab65797; Abcam), caveolin-1 (1:500 ab17052; Abcam), caveolin-1 (C3237; Sigma-Aldrich), and NCX (R3F1; Swant). Combinations of secondary anti-rabbit or anti-mouse antibodies of PLA PLUS and MINUS probes were used followed by hybridization, ligation, and amplification steps. Colocalization signals (proteins located within 40 nm of each other) were visualized with a standard Zeiss LSM710 upright laser-scanning confocal microscope.
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