TITLE: Indirect Proximity Ligation Assay (PLA) - Brightfield
AUTHORS: Ayse Ulusoy, Rita Pinto-Costa, Angela Rollar, Donato Di Monte

[DESCRIPTION]
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]
SECTION: Day 1
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)

SECTION: Day 1
2. Wash 2x5 min with Tris-HCl

SECTION: Day 1
3. Peroxidase quenching with BloxAll solution

use 300 µLand incubate for 30min


SECTION: Day 1
4. Wash 3x5 min with wash buffer A (see materials)


SECTION: Day 1
5. Antigen-retrieval with citrate buffer (+tween, pH = 6) at 95°C for 4 min
Note: Heat the solution to 95°C before adding on the samples



SECTION: Day 1
6. Wash 3x5 min with wash buffer A


SECTION: Day 1
7. Blocking: Incubate in Duolink Blocking Solution (300 µL)at 37°C for 60 min

SECTION: Day 1
8. Primary Antibody Incubation: Dilute antibodies in Duolink Antibody Diluent (200 µLµl/tube) and incubate 5 min 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 


SECTION: Day 2
9. Wash 3x10 minwith wash buffer A

SECTION: Day 2
10. 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 90 min

SECTION: Day 2
11. Wash 3x10 min with wash buffer A


SECTION: Day 2
12. 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 75 min



SECTION: Day 2
13. Wash 3x5 min with wash buffer A


SECTION: Day 2
14. 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 135 min 


SECTION: Day 2
16. Wash 2x5 min with wash buffer A 
Wash 1x5 min with Tris-buffered saline (no detergent)
SECTION: Day 2
15. Detection:
Dilute the Duolink Detection Brightfield Stock 1:5 in high-purity water 
add solution to the sections and incubate at RT for 70 min


SECTION: Day 2
17. 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) 
SECTION: Day 2
18. Wash 3x5 min with wash Tris-buffered saline


SECTION: Day 2
19. Mount the samples on slides and let them air-dry


SECTION: Day 2
20. Nuclear staining (optional): Incubate for 1 min 
Wash the staining off with running tap water for 10 min 




SECTION: Day 2
21. Dehydrate of samples in 1min each: 70% ethanol, 95% ethanol, 2x 100% ethanol 2x xylene


SECTION: Day 2
22. Coverslip samples with Histomount mounting medium