Neurons were prepared as described above for immunochemistry and stained overnight with indicated primary antibodies. PLA assay was then performed according to the manufacturer’s protocol using Duolink® PLA reagents from Sigma-Aldrich (Duolink® In Situ PLA® Probe Anti-Mouse PLUS Affinity purified Donkey anti-Mouse IgG (H+L), Duolink® In Situ PLA® Probe Anti-Rabbit PLUS Affinity purified Donkey anti-Rabbit IgG (H+L), Duolink® In Situ Detection Reagents Green). Negative controls using each primary antibody singly were used to determine non-specific PLA signal from each antibody. Neurons were imaged with a C2+ Nikon confocal microscope as described above, and PLA puncta were counted in ImageJ and normalized to number of cell bodies in each imaging field based on DAPI staining.
Duolink in situ pla probe anti mouse plus affinity purified donkey anti mouse igg h l
Manufactured by Merck Group
Duolink® In Situ PLA® Probe Anti-Mouse PLUS Affinity purified Donkey anti-Mouse IgG (H+L) is a laboratory reagent used for the detection and visualization of protein-protein interactions in situ. It is a secondary antibody conjugated with a DNA probe that can generate a signal when in close proximity to another Duolink® PLA® probe.
Automatically generated - may contain errors
3 protocols using duolink in situ pla probe anti mouse plus affinity purified donkey anti mouse igg h l
1
Proximity Ligation Assay for Neurons
2
Proximity Ligation Assay for Neurons
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Neurons were prepared as described above for immunochemistry and stained overnight with indicated primary antibodies. PLA assay was then performed according to the manufacturer’s protocol using Duolink® PLA reagents from Sigma-Aldrich (Duolink® In Situ PLA® Probe Anti-Mouse PLUS Affinity purified Donkey anti-Mouse IgG (H+L), Duolink® In Situ PLA® Probe Anti-Rabbit PLUS Affinity purified Donkey anti-Rabbit IgG (H+L), Duolink® In Situ Detection Reagents Green). Negative controls using each primary antibody singly were used to determine non-specific PLA signal from each antibody. Neurons were imaged with a C2+ Nikon confocal microscope as described above, and PLA puncta were counted in ImageJ and normalized to number of cell bodies in each imaging field based on DAPI staining.
3
Proximity Ligation Assay for Protein-Protein Interactions
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HUVECs were fixed in 4% formaldehyde and incubated with antibodies (anti-MEF2A [mouse origin], Smad2 [rabbit origin]) at 4°C overnight. The cells were subsequently incubated with the PLUS and MINUS PLA probes (Duolink® In Situ PLA® Probe Anti-Mouse PLUS Affinity-purified Donkey anti-Mouse IgG (H+L), Sigma, DUO92001; Duolink® In Situ PLA® Probe Anti-Rabbit PLUS Affinity-purified Donkey anti-Rabbit IgG (H+L), Sigma DUO92002) at 37°C for 1h. Following incubation, the cells were washed and incubated in the Ligation-Ligase solution for 30 min at 37°C. Following an additional wash, the cells were incubated in Amplification-Polymerase solution for 100 min at 37°C. The samples were mounted using Duolink In Situ Mounting Medium with DAPI (Sigma, DUO82040), allowed to adhere for 15 min, and analyzed using a confocal microscope.
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