Nov 12, 2023
Proximity Ligation Assay (PLA)
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Proximity Ligation Assay (PLA). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko6ydv5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90825
Abstract
Proximity Ligation Assay (PLA)
The PLA assay was performed as described previously with minor modifications 48. The following antibodies were used for the PLA experiments: Syn 204 against h-αSyn (Santa Cruz Biotechnology Cat# sc-32280, RRID:AB_628319)(1:100) and EPR12790 against VAMP-2 (Abcam Cat#
ab214590)(1:300).
The in-situ PLA was performed on fixed primary neurons
with DuoLink PLA technology probes and reagents (Sigma-Aldrich Cat# DUO92002,
DUO92004, DUO82049, DUO92008, and DUO92014), following the manufacturer’s
protocol. First, the neurons were permeabilized with PBS + 0.4% Triton X-100
for 10 min.
After two PBS washes, the cells were incubated with a blocking
solution for 2 hours at 37 °C and then incubated with the primary antibodies
for 30 min at room temperature.
The coverslips were washed twice for 5 min with
buffer A, followed by incubation with the PLA probes (secondary antibodies
against two different species bound to two oligonucleotides: anti-mouse MINUS (Sigma-Aldrich Cat#
DUO92004 (also DUO92004-30RXN, DUO92004-100RXN), RRID:AB_2713942) and anti-rabbit PLUS) (Bethyl Cat# OLK-92002-0100,
RRID:AB_10950581)in antibody diluent for 30 min at 37 °C. After two washes of 5
min with buffer A, the ligation step was performed with ligase diluted in
ligation stock for 30 min at 37 °C.
The coverslips were washed with buffer A
twice for 2 min before incubation for 50 min with amplification stock solution
at 37 °C. After two washes of 10 min with buffer B. Finally, the coverslips
were washed with PBS and mounted with Duolink in situ mounting medium
(Sigma-Aldrich Cat# DUO82040-5 ML).
A negative control experiment was performed
for every antibody, where only one antibody was incubated with the PLA probes.
The experiments were performed 2 times. The experiments were performed 2 times.
Average signal intensities were measured using the MetaMorph Microscopy Automation and Image
Analysis Software (RRID:SCR_002368) (https://www.moleculardevices.com/products/cellular-imaging-systems/acquisition-and-analysis-software/metamorph-microscopy#gref) and plotted using GraphPad Prism
software [(RRID:SCR_002798) http://www.graphpad.com/].