Duo92007 100rxn

PLA (DUO92004-100RXN; DUO92002-100RXN; DUO92007-100RXN, Sigma-Aldrich) was performed in paraffin-embedded fixed tissue prepared for immunofluorescence, as described above. To detect neurons in brain, an immunofluorescence staining for MAP2 was performed before PLA, then samples were incubated with specific primary antibodies to the proteins of interest for PLA. Secondary antibodies conjugated with oligonucleotides were added to the reaction and incubated. Ligation solution, consisting of two oligonucleotides and ligase, was added. In this assay, the oligonucleotides hybridize to the two proximity ligation probes and join to a closed loop if they are in close proximity. Amplification solution, consisting of nucleotides and fluorescently labeled oligonucleotides, were added together with polymerase. The oligonucleotide arm of one of the proximity ligation probes acts as a primer for “rolling-circle amplification” using the ligated circle as a template, and this generates a concatemeric product. Fluorescently labeled oligonucleotides hybridize to the rolling circle amplification product. The proximity ligation signal was visible as a distinct fluorescent spot and was analyzed by confocal microscopy. Control experiments were run in parrallel by primary antibodies deletion. For more details and step-by-step protocol see(Keeney et al., 2021 (link)).