Proximity ligation assay (PLA) was performed using the anti-Mouse MINUS probe, anti-Rabbit PLUS probe and the orange detection reagent (Sigma-Aldrich) as previously described(53 (link)). Actin was visualized using actistain 670 and imaged on a Leica scanning confocal (SP5) with a HCX Plan Apochromat 63×/1.40–0.60 NA oil λ blue objective. The total number of distinct PLA spots per cell were quantified using the threshold function of ImageJ.
Orange detection reagent
Manufactured by Merck Group
The Orange detection reagent is a laboratory product designed to detect the presence of orange-colored compounds in samples. It is a chemical solution that, when added to a sample, will change color or produce a visible reaction in the presence of orange-colored substances. The core function of this reagent is to serve as a detection tool for identifying and analyzing orange-colored materials in a variety of laboratory applications.
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3 protocols using orange detection reagent
1
Proximity Ligation Assay for Protein Interactions
2
Proximity Ligation Assay for Protein Interactions
3
Proximity Ligation Assay for LMTK2 and p35
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Proximity ligation assays (PLAs) were performed with rabbit anti-LMTK2 and goat anti-KLC1, and with rabbit anti-p35 and goat anti-KLC1 antibodies using Duolink PLA probes and orange detection reagent (Sigma) according the manufacturer’s protocol. Briefly, cells were fixed and permeabilised as described above for immunofluorescence staining, blocked with PLA blocking buffer for 30 min at 37 °C and incubated with primary antibodies for 1 h. Following washing, samples were incubated with PLA probes for 1 h at 37 °C and after further washing, probes were ligated for 30 min at 37 °C and amplified for 100 min at 37 °C. Following PLAs, cells were incubated with tubulin primary antibody in PBS at 4 °C for 16 h, washed with PBS and incubated with fluorescent conjugated secondary antibody for 1 h in PBS. Following further washing, samples were mounted as described above. Control reactions to demonstrate the specificity of the assays involved omission of primary antibodies. Signals were quantified using ImageJ particle analysis function as described [42 (link)].
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