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[Home](https://www.sigmaaldrich.com/US/en)[Protein Expression](https://www.sigmaaldrich.com/US/en/applications/protein-biology/protein-expression)Duolink® *In Situ* Short Instructions - Fluorescence
# Duolink® *In Situ* Short Instructions - Fluorescence
1.__Blocking__
Add blocking solution to samples.
Incubate.
• Remove block
2.__Primary Antibodies__
Dilute the primary antibodies in appropriate buffer and apply to samples.
Incubate.
• Wash in suitable buffer for 2 × 5 min
3.__PLA® Probes__
Dilute the two PLA probes 1:5 in appropriate buffer and apply to samples.
Incubate for 60 min at +37 °C.
• Wash in 1x Wash Buffer A for 2 × 5 min
4.__Ligation__
Dilute the Ligation stock 1:5 in H2O.
Dilute the Ligase at 1:40 in the solution and apply the mix to samples.
Incubate for 30 min at +37 °C.
• Wash in 1x Wash Buffer A for 2 × 2 min
5.__Amplification__
Dilute the Amplification stock 1:5 in H2O. Dilute the Polymerase at 1:80 in the solution and apply the mix to samples.
Incubate for 100 min at +37 °C.
• Wash in 1x Wash Buffer B for 2 × 10 min
• Wash in 0.01x Wash Buffer B for 1 min
6.__Preparation for Imaging__
Mount the samples with Duolink In Situ Mounting Medium with DAPI, wait for 15 min, and analyze in a fluorescence or confocal microscope.
## Materials
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__Notes__:
- Use open droplet reactions without a cover slip and perform all incubations in a humidity chamber.
- Use a freezing block when removing the enzymes from the freezer (-20 ºC).
- Washing should be done in a minimum volume of 70 mL on a shaker with gentle orbital shaking.
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- [Creating PLA® Probes](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/creating-pla-probes-duolink-application-note)
- [Using Duolink® in Protein Interaction Studies](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink)
- [Duolink® PLA Brightfield Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink-brightfield-user-manual)
- [Counterstaining after the Duolink *In Situ* Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink-counterstain)
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- [Duolink® PLA Multicolor Detection Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink-pla-multicolor-detection-protocol)
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- [Duolink® Proximity Ligation Assay](https://www.sigmaaldrich.com/US/en/products/protein-biology/duolink-proximity-ligation-assay)
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