Indirect Proximity Ligation Assay (PLA) - Fluoresence
Ayse Ulusoy, Rita Pinto-Costa, Angela Rollar, Donato Di Monte
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.
Steps
Day 1
Pick 35um cut brain sections and transfer them to 1.5mL Eppendorf tubes:
Note: all incubation and wash steps are performed by shaking Eppendorf tubes at 250rpm (e.g., thermomixer)
Wash 2x0h 5m 0s with Tris-HCl
Wash 3x0h 5m 0s with wash buffer A (see materials)
Antigen-retrieval with citrate buffer (+tween, pH = 6) at 95°C for 0h 4m 0s
Note: Heat the solution to 95°C before adding on the samples
Wash 3x0h 5m 0s with wash buffer A
Blocking: Incubate in Duolink Blocking Solution (300µL)at 37°C for 1h 0m 0s
Primary Antibody Incubation : Dilute antibodies in Duolink Antibody Diluent (200µL) and incubate 0h 5m 0s 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
Day 2
Wash 3x0h 10m 0swith wash buffer A
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 for1h 30m 0s
Wash 3x0h 10m 0s with wash buffer A
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 1h 15m 0s
Wash 3x0h 5m 0s with wash buffer A
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 2h 30m 0s
Wash 2x0h 5m 0s with wash buffer B
Wash 1x0h 1m 0s with wash 0.1x buffer B
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
