Detection of Protein-Protein Interactions in Drosophila Larvae Using a Proximity Ligation Assay

Abstract

Source: Wang, S., et al. Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay. J. Vis. Exp. (2015).

This video demonstrates the visualization of protein-protein interactions at the Drosophila neuromuscular junction (NMJ) using Proximity Ligation Assay (PLA). After isolating the body walls, interacting proteins are probed with primary antibodies and PLA-conjugated secondary antibodies. The proximity of the interacting proteins facilitates the ligation of probes with connector oligonucleotides, forming circular DNA, which is then amplified by rolling circle amplification. Fluorescent probes bind to the amplified DNA, allowing the visualization of protein-protein interactions at the NMJ.

Protocol

1. Body Wall Preparation

NOTE: Preparation of third instar larval body walls (for study of the NMJs which innervate the body wall muscles) was performed as previously described but with some modifications.

1. Dissection
   1. Raise fly stocks and crosses at 25 °C for five to six days using standard procedures.
   2. Pick crawling third instar larvae from vials or bottles using fine forceps.
   3. Wash the larvae in a small Petri dish containing Phosphate Buffer Saline (PBS) to remove any food particles.
   4. Place a single larva onto a sylgard disc and immerse it in a few....

Results

Figure 1: Preparation of third instar larval body walls. (A-F) Schematic drawings representing a body wall dissection. (A) Place a single larva onto a sylgard disc with its dorsal side facing up so that the two tracheal tracts are visible. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Forceps (fine #5)	Almedic	A10-704
Sylgard Disc	World Precision Instruments	SYLG184	Mix elastomer base and curing agent in a 10:1 ratio. Set for 30 min. Pour into a mold (e.g. use a 12-well cell culture plate). Let cure for at least 24 hr. Adhere to the lid of a 60x15mm petri dish lid when dissecting.
Minutien Pins (0.0125 mm tip diameter)	Fine Science Tools	26002-10
Microdissection Scissors (ultra fine)	Fine Science Tools	15200-00
Platform Slides			Glue two 22x22mm coverslips onto a microscope slide with clear nail polish, leaving a <20mm gap in between for sample mounting.
w1118	Bloomington Drosophila Stock Center	3605
hts01103	Bloomington Drosophila Stock Center	10989	Stock was re-balanced over a GFP balancer so that homozygous mutants can be selected based on the absence of GFP signal.
1x PBS (Phosphate Buffered Saline): 3mM NaH2PO4, 7mM Na2HPO4, 130mM NaCl, pH 7.0			NaH2PO4 (Caledon Laboratories - 8180-1), Na2HPO4 (Caledon - 8120-1), NaCl (Caledon - 7560-1)
Bouin's Solution	Sigma-Aldrich	HT10132
4% PFA (Paraformaldehyde): 4% PFA in 1x PBS			PFA (Anachemia Science - 66194-300). See doi:10.1101/pdb.rec9959 Cold Spring Harb Protoc 2006 for instructions on how to make the solution.
1x PBT (Phosphate Buffered Saline with Triton): 1x PBS with 0.01% Triton			Triton X-100 (Sigma-Aldrich - T8787)
1% BSA (Bovine Serum Albumin): 1% BSA in 1x PBT			BSA (Bioshop Canada - ALB001). Store at 4 °C.
mouse anti-Dlg (Discs large)	Developmental Studies Hybridoma Bank	4F3	Use at a 1:10 dilution in 1% BSA.
rabbit anti-HtsM (Hu-li tai shao)			Provided by Dr. Lynn Cooley (Yale University). Use at a 1:250 dilution in 1% BSA.
rabbit anti-Pak (p21-activated kinase)			Provided by Dr. Nicholas Harden (Simon Fraser University). Use at a 1:500 dilution in 1% BSA.
mouse anti-Wg (Wingless)	Developmental Studies Hybridoma Bank	4D4	Use at a 1:5 dilution in 1% BSA.
goat anti-Hrp (Horseradish peroxidase)	Jackson ImmunoResearch	123-065-021	Use at a 1:200 dilution in 1% BSA.
FITC-conjugated donkey anti-goat	Jackson ImmunoResearch	705-095-003	Use at a 1:200 dilution in 1% BSA.
Duolink In Situ PLA Probe anti-mouse MINUS	Sigma-Aldrich	DUO92004
Duolink In Situ PLA Probe anti-rabbit PLUS	Sigma-Aldrich	DUO92002
Duolink In Situ Detection Reagents Red	Sigma-Aldrich	DUO92008
1x Wash Buffer A: 0.01M Tris, 0.15M NaCl, 0.05% Tween 20, pH 7.4	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1), Tween 20 (Fisher Scientific - BP337)
1x Wash Buffer B: 0.2M Tris, 0.1M NaCl, pH 7.5	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)
0.01x Wash Buffer B: 2mM Tris, 1mM NaCl, pH 7.5	Sigma-Aldrich	DUO82049	Tris (Caledon Laboratories - 8980-1), NaCl (Caledon - 7560-1)
Duolink In Situ Mounting Medium with DAPI	Sigma-Aldrich	DUO82040