Description_x000D_ Application_x000D_ 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._x000D_ Follow the Duolink™ In Situ Brightfield Protocol to use this product._x000D_ Visit our Duolink™ PLA Resource Center for information on how to run a Duolink™ experiment, applications, troubleshooting, and more._x000D_ 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._x000D_ Specificity_x000D_ Brightfield detection reagents are used for samples with significant autofluorescence._x000D_ Application Note_x000D_ 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._x000D_ Let us do the work for you, learn more about our Custom Service Program to accelerate your Duolink™ projects_x000D_ View full Duolink™ product list_x000D_ Features and Benefits_x000D_ • No overexpression or genetic manipulation required_x000D_ • High specificity (fewer false positives)_x000D_ • Single molecule sensitivity due to rolling circle amplification_x000D_ • Relative quantification possible_x000D_ • No special equipment needed_x000D_ • Quicker and simpler than FRET_x000D_ • Increased accuracy compared to co-IP_x000D_ • Publication-ready results_x000D_ Components_x000D_ This product is comprised of the following:_x000D_ Box A:_x000D_ • 5x Ligation - Contains oligonucleotides that hybridize to the PLA probes and all components needed for ligation except the Ligase_x000D_ • 1x Ligase (1 unit/μL)_x000D_ • 5x Amplification - Contains all components needed for Rolling Circle Amplification (RCA) except the Polymerase_x000D_ • 5x Detection Brightfield - Contains oligonucleotide probes labeled with horseradish peroxidase (HRP) that hybridize to the RCA product_x000D_ • 1x Polymerase (10 units/μL)_x000D_ Box B:_x000D_ • 1x Hydrogen Peroxide - Contains 0.3% hydrogen peroxide_x000D_ • Substrate Reagents A-D - Contains all substrate components needed for HRP enzymatic reaction_x000D_ • 1x Nuclear Stain - Contains Mayer’s hematoxylin solution for cell nuclei staining_x000D_ See datasheet for more information._x000D_ Not included in Detection kit:_x000D_ Primary antibodies, PLA probes, wash buffers, mounting medium_x000D_ Other Notes_x000D_ Storage/Stability_x000D_ 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._x000D_ Store Box B and all of its components at 2-8°C._x000D_ Legal Information_x000D_ Duolink is a trademark of Sigma-Aldrich Co. LLC_x000D_ PLA is a registered trademark of Sigma-Aldrich Co. LLC