PLA was performed using Duolink In Situ PLA Reagents Red (Sigma-Aldrich, Cat#DUO92008) according to the manufacturer’s protocol. Ramos cells were attached to coverslips with 0.1% poly-L-lysine (Sigma-Aldrich, Cat#P8920) for 1 h at RT. Cells were permeabilized, fixed with methanol for 10 min at −20 °C, blocked with Duolink blocking solution, and incubated with primary antibodies. PLA Probe Anti-Mouse MINUS (Sigma-Aldrich) and PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich) were used as secondary probes. Samples were mounted with DAPI. PLA signals were detected using a Nikon Ti Microscope (λex = 594 nm; λem = 624 nm). Images were taken with a Nikon DS-Qi2 camera. PLA foci were counted automatically with ImageJ [57 (link)] after threshold adjustment using function “analyze particles”.
Duolink in situ pla reagents red
Manufactured by Merck Group
Duolink In Situ PLA Reagents Red is a laboratory product manufactured by Merck Group. It is used for in-situ proximity ligation assay (PLA) to detect and visualize protein-protein interactions in cells or tissue samples.
Automatically generated - may contain errors
2 protocols using duolink in situ pla reagents red
1
Proximity Ligation Assay Protocol
2
Proximity Ligation Assay in Ramos Cells
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PLA was performed using Duolink In Situ PLA Reagents Red (Sigma-Aldrich, Cat#DUO92008) according to the manufacturer's protocol. Ramos cells were attached to coverslips with 0.1 % poly-L-lysine (Sigma-Aldrich, Cat#P8920) for 1 h at RT. Cells were permeabilized, fixed with methanol for 10 min at -20 °C, blocked with Duolink blocking solution, and incubated with primary antibodies. PLA Probe Anti-Mouse MINUS (Sigma-Aldrich) and PLA Probe Anti-Rabbit PLUS (Sigma-Aldrich) were used as secondary probes. Samples were mounted with DAPI. PLA signals were detected using a Nikon Ti Microscope (λex=594 nm; λem=624 nm). Images were taken with a Nikon DS-Qi2 camera. PLA foci were counted automatically with ImageJ (57) after threshold adjustment using function "analyze particles".
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