Aug 02, 2023
Indirect Proximity Ligation Assay (PLA) - Brightfield
- Ayse Ulusoy1,
- Rita Pinto-Costa1,
- Angela Rollar1,
- Donato Di Monte1
- 1DZNE
Protocol Citation: Ayse Ulusoy, Rita Pinto-Costa, Angela Rollar, Donato Di Monte 2023. Indirect Proximity Ligation Assay (PLA) - Brightfield. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x94klqe/v1
Manuscript citation:
doi: 10.1126/sciadv.abn0356
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: August 02, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85848
Keywords: post-translational modification, alpha-synuclein, oxidative stress, ASAPCRN, brightfield indirect proximity ligation assay, indirect proximity ligation assay, nitration of mitochondrial enzyme, nitration of protein, mitochondrial enzyme, synuclein, protein modification, dependent ligation, protein, protein interaction, subunit ndufb8, protein complex formations within cell, nitrated alpha, powerful molecular technique, specific antibody, pla protocol
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000420
Abstract
Indirect Proximity Ligation Assay (PLA) is a powerful molecular technique used to detect and visualize protein-protein interactions, protein modifications, and protein complex formations within cells or tissues. This method is based on the principles of proximity-dependent ligation and utilizes specific antibodies to detect the nitration of proteins on free-floating brain sections. Here we describe the PLA protocol that we routinely use in our laboratory to detect nitrated alpha-synuclein and nitration of mitochondrial enzymes such as SOD2 and the mitochondrial complex 1 subunit NDUFB8.
Materials
Duolink InSitu PLA probe anti-rabbit PLUS kit: DUO92002-100RXN
Duolink InSitu PLA probe anti-mouse MINUS kit: DUO92004-100RXN
Duolink InSitu Detection Reagents Brightfield kit: DUO92012-100RXN
Duolink wash buffer A: DUO82047-20L
Histomount mounting medium: Life Technologies 008030
Antibodies:
mouse anti-3-NT: 1:250; ab61392, Abcam
rabbit anti-human alpha-synuclein (clone MJFR1): 1:4000, ab138501, Abcam
rabbit anti-SOD1: 1:1000; ADI-SOD-110, Enzo Life Sciences
rabbit anti-NDUFB8: 1:300; 14794, Proteintech
Troubleshooting
Day 1
Pick 35um cut brain sections and transfer them to 1.5 mL Eppendorf tubes
Note: all incubation and wash steps are performed by shaking Eppendorf tubes at 250rpm (e.g., thermomixer)
Wash 2x00:05:00 with Tris-HCl
5m
Peroxidase quenching with BloxAll solution
use 300 µL and incubate for 30min
Wash 3x00:05:00 with wash buffer A (see materials)
5m
Antigen-retrieval with citrate buffer (+tween, pH = 6) at 95°C for 00:04:00
Note: Heat the solution to 95°C before adding on the samples
4m
Wash 3x00:05:00 with wash buffer A
5m
Blocking: Incubate in Duolink Blocking Solution (300 µL )at 37°C for 01:00:00
1h
Primary Antibody Incubation: Dilute antibodies in Duolink Antibody Diluent (200 µL µl/tube) and incubate Overnight room temperature.
for nitrated alpha-synuclein: mouse anti-3-NT and rabbit anti-h-alpha-synuclein
for nitrated SOD2: mouse anti-3-NT and rabbit anti-SOD2
for nitrated NDUFB8: mouse anti-3-NT and rabbit anti-NDUFB8
see materials for dilutions and catalog numbers
5m
Day 2
10m
Wash 3x00:10:00 with wash buffer A
10m
PLA probe incubation:
Prepare anti-mouse MINUS and anti-rabbit PLUS probes following manufacturer's instructions, add solution on sections and incubate at 37°C for 01:30:00
1h 30m
Wash 3x00:10:00 with wash buffer A
10m
Ligation:
Dilute the Duolink Ligation Buffer 1:5 in high-purity water
Add the ligase (diluted 1:40) just before incubation (keep cold on freezer block!)
add the ligation solution on sections and incubate at 37°C for 01:15:00
1h 15m
Wash 3x00:05:00 with wash buffer A
5m
Amplification:
Dilute the Duolink Amplification Buffer 1:5 in high-purity water
Add the polymerase (diluted 1:80) just before incubation (keep cold on freezer block!)
Add the amplification solution to the sections and incubate at 37°C for 02:15:00
2h 15m
Detection:
Dilute the Duolink Detection Brightfield Stock 1:5 in high-purity water
add solution to the sections and incubate at RT for 01:10:00
1h 10m
Wash 2x00:05:00 with wash buffer A
Wash 1x00:05:00 with Tris-buffered saline (no detergent)
10m
Developing:
Dilute the Duolink Brightfield Substrate solutions in high-purity water:
§ Substrate A (1:70)
§ Substrate B (1:100)
§ Substrate C (1:100)
§ Substrate D (1:50)
Incubate sections at RT for 1-5min (depending on desired intensity outcome)
Wash 3x00:05:00 with wash Tris-buffered saline
5m
Mount the samples on slides and let them air-dry
Nuclear staining (optional): Incubate for 00:01:00
Wash the staining off with running tap water for 00:10:00
11m
Dehydrate of samples in 1min each: 70% ethanol, 95% ethanol, 2x 100% ethanol 2x xylene
Coverslip samples with Histomount mounting medium
Protocol references
doi: 10.1126/sciadv.abn0356