This protocol describes the application of proximity ligation assay for detection of Smad proteins and the interactions between Smads and STAT3 or transcriptional cofactors in floating or adherent cells.
Method Article
Detecting Smad-STAT networks using proximity ligation assay
https://doi.org/10.1038/protex.2016.026
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This protocol describes the application of proximity ligation assay for detection of Smad proteins and the interactions between Smads and STAT3 or transcriptional cofactors in floating or adherent cells.
• Duolink® In Situ Detection Reagents Red (Sigma-Aldrich, DUO92008): Ligation (5x), Ligase (1 U/μl), Amplification (5x), Polymerase (10 U/μl)
• Duolink PLA Probes:
Duolink® In Situ PLA Anti-Mouse PLUS Affinity purified Donkey anti-Mouse IgG (H+L) (Sigma-Aldrich, DUO92001)
Duolink® In Situ PLA Anti-Rabbit PLUS Affinity purified Donkey anti-Rabbit IgG (H+L) (Sigma-Aldrich, DUO92002)
Duolink® In Situ PLA Anti-Mouse MINUS Affinity purified Donkey anti-Mouse IgG (H+L) (Sigma-Aldrich, DUO92004)
Duolink® In Situ PLA Anti-Rabbit MINUS Affinity purified Donkey anti-Rabbit IgG (H+L) (Sigma-Aldrich, DUO92005)
Duolink Blocking solution
Duolink Antibody diluent
• Fixation buffer (4% formaldehyde in PBS, store at room temperature (RT))
• Permeabilization buffer (0.1% Triton X-100 in PBS, store at 4° C )
• Primary antibodies (cf. Table 1, 2)
• PBS, 2% FBS PBS
• Wash Solution (Dissolve 8.8 g NaCl, 1.2 g Tris base and 0.5 ml Tween 20 in 800 ml high purity water. Adjust pH to 7.4 using HCl. Add high purity water to 1000 ml (final concentrations: 0.01 M Tris, 0.15 M NaCl and 0.05% Tween 20). Filter the solution through a 0.22 μm filter and store at 4° C. Bring the solutions to room temperature before use.)
• Duolink In Situ Mounting Medium with DAPI (Sigma-Aldrich, DUO82040)
• Water-proof pen (Dako Pen, Dako, S2002)
• Poly-L-Lysine coated slide glass (0.01% Poly-L-Lysine solution, Sigma-Aldrich, P8920) and cover slip
• High purity water
• Confocal microscopy (e.g. Carl Zeiss, LSM700)
• Cytospin (for floating cells, e.g. Thermo Scientific Cytospin 4 Cytocentrifuge, Hanil Cellspin)
• Humidity chamber
• Incubator (37° C )
• Centrifuge, 1500 rpm for 10 min at 4° C.
• Remove the supernatant carefully and wash cells with 2% FBS PBS by centrifuge, 1500 rpm for 10 min at 4° C.
• Remove the supernatant and wash again with PBS by centrifuge, 1500 rpm for 10 min at 4° C.
Adherent cells such as dendritic cells or cancer cells are cultured on the coverslip in the culture dishes.
• Wash the cells twice with PBS (1 ml in microcentrifuge tubes volume 1.5 ml) by centrifuge, 1500 rpm for 10 min at RT.
• Permeabilize the cells with permeabilization buffer (500 μl) for 15 min at RT.
• Wash the cells twice with PBS by centrifuge, 1500 rpm for 10 min at RT.
• Prepare thin-layers of the fixed cells on the Poly-L-Lysine coated slides by centrifugation using cytospin (1~2x105 cells/slide, 800 rpm for 10 min at RT).
• After cytospin centrifugation, draw a circle around the samples by water-proof pen such as Dako Pen.
Blocking
• Block the slides by blocking solution for 30 min at 37° C in humidity chamber.
• Tap off the blocking solution carefully from the slides without wash.
Incubation with primary antibodies
• Incubate the slides immediately with primary antibodies diluted by the antibody diluent (1:50, Table 1 or 2) overnight at 4° C in humidity chamber.
(Cell surface marker staining if needed)
• (Incubate the slides with the primary rat anti-mouse antibody to detect cell surface markers (e.g. rat anti-mouse CD8a antibody, Abcam ab22378) diluted by the antibody diluent (1:50) overnight at 4°C in humidity chamber.)
Day 2
Incubation with PLA probes
• Tap off the primary antibody solution from the slides, and wash the slides twice carefully for 5 min by the wash solution.
• Incubate the slides with the PLA probes diluted by the antibody diluent (Table 1 or 2) for 1 hr at 37° C in humidity chamber.
• Wash the slides twice carefully for 5 min by the wash solution.
Ligation
• Add the Ligation-Ligase solution diluted by high purity water and incubate the slides for 30 min at 37° C in humidity chamber.
• Wash the slides twice carefully for 5 min by the wash solution.
Amplification
• Add the Amplification-Polymerase solution diluted by high purity water and incubate the slides for 100 min at 37° C in the dark humidity chamber.
• Wash the slides twice carefully for 5 min by the wash solution and remove the remained wash solution completely using a pipette.
(Cell surface marker staining if needed)
• (Incubate the slides with the secondary goat anti-rat IgG (H+L) antibody Alexa Fluor® 488 (ThermoFisher SCIENTIFIC, A-11006) diluted by the antibody diluent (1:1000) for 1 hr at RT in the dark humidity chamber.)
• (Wash the slides twice carefully for 5 min by the wash solution and remove the remained wash solution completely using a pipette.)
Nuclear staining and mounting
• Add the minimal volume of the Duolink In Situ Mounting Medium with DAPI for nucleus staining and mounting, and cover the slides with cover slips.
• Remove air bubbles from the slide by pushing the cover slips carefully.
• Store the slide at 4° C in the dark before acquiring the images using a confocal microscope.
• PLA signals are quantified using BlobFinder software (Centre for Image Analysis, Uppsala University, http://www.cb.uu.se/~amin/BlobFinder/).
Yoon, J.H. et al. Phosphorylation status determines the opposing functions of Smad2/Smad3 as STAT3 cofactors in TH17 differentiation. Nat Commun 6, 7600 \(2015). Duolink In Situ Fluorescence User Guide \(http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Instructions/1/duolink-fluorescence-user-manual.pdf).The authors declare no competing financial interests.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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