[Skip to Content](https://www.sigmaaldrich.com#main-content) [](https://www.sigmaaldrich.com/US/en) Products Cart0 USEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/US/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/US/en/quick-order) Cart0 [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 Sorry, an unexpected error has occurred Response not successful: Received status code 500 __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. __Related Articles__ - [Yeast Growth Methods](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/microbiological-testing/pathogen-and-spoilage-testing/yeast-growth-protocols) - [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) - [Duolink® PLA Flow Cytometry Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink-cytometry-protocol) - [Duolink® PLA Fluorescence Protocol](https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-and-nucleic-acid-interactions/duolink-fluorescence-user-manual) - [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) __Related Product Categories__ - [Duolink® Proximity Ligation Assay](https://www.sigmaaldrich.com/US/en/products/protein-biology/duolink-proximity-ligation-assay) - [Biological Buffers](https://www.sigmaaldrich.com/KR/en/products/chemistry-and-biochemicals/biochemicals/biological-buffers) Top __Sign In To Continue__ To continue reading please sign in or create an account. Sign In__Don't Have An Account?__Register An unknown error has occured. - English - EN - Español - ES [Learn More](https://www.sigmaaldrich.com)