Aug 02, 2023
Indirect Proximity Ligation Assay (PLA) - Fluoresence
- 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) - Fluoresence. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged36dv47/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: 85854
Keywords: post-translational modification, alpha-synuclein, oxidative stress, ASAPCRN
Funders Acknowledgement:
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
Blocking solution: Vector Lab SP-6000-100
Duolink In Situ Wash Buffers, Fluorescence: DUO82049-20L
Duolink InSitu PLA probe anti-rabbit PLUS kit: DUO92002-100RXN
Duolink InSitu PLA probe anti-mouse MINUS kit: DUO92004-100RXN
Duolink In Situ Detection Reagents Red: DUO92008-100RXN
Duolink In Situ Mounting Medium with DAPI: DUO82040-5ML
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
Day 1
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
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 ) 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
Day 2
10m
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: This step is light-sensitive! incubation is performed using aluminum foil to protect samples from light.
Dilute the Duolink Amplification Buffer (red) 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 02:30:00
2h 30m
Wash 2x00:05:00 with wash buffer B
Wash 1x00:01:00 with wash 0.1x buffer B
6m
Additional immunohistochemical staining (optional): if immunohistochemical counter-staining is going to be performed it is important to avoid the use of detergents in wash buffers and perform long incubations in cold.
Mount the samples on slides and let them air-dry for 5 mins
Coverslip samples with Duolink In Situ Mounting Medium with DAPI, store samples in the dark at 4°C
Protocol references
doi: 10.1126/sciadv.abn0356