Duolink(TM) In Situ Detection Reagents Brightfield (C15-1356-119)

Application

Duolink^®proximity ligation assay(PLA^®) allows for endogenous detection of protein interactions, post translational modifications, and protein expression levels at the single molecule level in fixed cells and tissue samples.

Follow the Duolink^® In Situ Brightfield Protocol to use this product.

Visit our Duolink^® PLA Resource Center for information on how to run a Duolink^® experiment, applications, troubleshooting, and more.

To perform a complete Duolink^® PLA in situ experiment you will need two primary antibodies (PLA, IHC, ICC or IF validated) that recognize two target epitopes. Other necessary reagents include a pair of PLA probes from different species (one PLUS and one MINUS), detection reagents, wash buffers, and mounting medium. Note that the primary antibodies must come from the same species as the Duolink^® PLA probes. Analysis is carried out using HRP for brightfield detection.

Specificity
Brightfield detection reagents are used for samples with significant autofluorescence.

Application Note
Two primary antibodies raised in different species are needed. Test your primary antibodies (IgG-class, mono- or polyclonal) in a standard immunofluorescence (IF), immunohistochemistry (IHC) or immunocytochemistry (ICC) assay to determine the optimal fixation, blocking, and titer conditions. Duolink^® in situ reagents are suitable for use on fixed cells, cytospin cells, cells grown on slide, formalin-fixed, paraffin embedded (FFPE), or tissue (fresh or frozen). No minimum number of cells is required.

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Features and Benefits

• No overexpression or genetic manipulation required
• High specificity (fewer false positives)
• Single molecule sensitivity due to rolling circle amplification
• Relative quantification possible
• No special equipment needed
• Quicker and simpler than FRET
• Increased accuracy compared to co-IP
• Publication-ready results

Components

This product is comprised of the following:
Box A:

• 5x Ligation - Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase
• 1x Ligase (1 unit/μL)
• 5x Amplification - Contains all components needed for Rolling Circle Amplification (RCA) except the Polymerase
• 5x Detection Brightfield - Contains oligonucleotide probes labeled with horseradish peroxidase (HRP) that hybridize to the RCA product
• 1x Polymerase (10 units/μL)

Box B:

• 1x Hydrogen Peroxide - Contains 0.3% hydrogen peroxide
• Substrate Reagents A-D - Contains all substrate components needed for HRP enzymatic reaction
• 1x Nuclear Stain - Contains Mayer′s hematoxylin solution for cell nuclei staining

See datasheet for more information.

Not included in Detection kit:

Primary antibodies, PLA probes, wash buffers, mounting medium

Other Notes

Storage/Stability
Store Box A and all of its components at -20°C. The enzymes should be kept cold (-20°C) at all times; Use a freezing block when removing them from the freezer.
Store Box B and all of its components at 2-8°C.

Legal Information

Duolink is a registered trademark of Sigma-Aldrich Co. LLC

PLA is a registered trademark of Sigma-Aldrich Co. LLC