Duolink® PLA is an innovative technology that combines the use of antibodies with rolling circle amplification, to take immunodetection to the next level. This video will show you how to perform a Duolink® PLA experiment with cells on a slide that have already been fixed and permeabilized by the user.
To successfully run a Duolink® proximity ligation assay, you will need the following products to perform your immunodetection: A pair of PLA probes, both PLUS and MINUS that match to the host of your primary antibodies; Detection Reagents; Wash Buffers; and Mounting Medium.
You will also need a hydrophobic pen, humidity chamber, heat transfer block, freezer block, staining jar and a shaker. Your sample must first be fixed and permeabilized on the slide before you can begin.
When you are ready, tap off excess wash buffer and use a hydrophobic pen, or aspirate, around the wells. Add one drop of blocking solution per well and incubate in a pre-heated humidity chamber, or heat transfer block, at thirty-seven degrees Celsius for one hour.
During the one-hour incubation, dilute the primary antibodies in antibody diluent. Primary antibodies need to have been raised in either mouse, rabbit or goat in order to be recognized by the Duolink® PLA probes.
After incubation is complete, tap off blocking solution and add the primary antibody. Incubate at the optimized temperature and time. After incubation, wash the slide twice for five minutes each in Wash Buffer A with gentle orbital shaking at room temperature.
During washing, dilute the two Duolink® PLA probes together 1:5 in antibody diluent. Tap off excess wash buffer and add the PLA probe solution to each well. Incubate for one hour at thirty-seven degrees Celsius.
After incubation, wash the slide twice for five minutes each in Wash Buffer A with gentle orbital shaking at room temperature. During washing, make 1x ligation buffer by diluting the 5x ligation buffer 1:5 in high purity water. Make sure the 5x ligation buffer is completely thawed with no visible precipitate prior to dilution.
Dilute the 1x ligase enzyme 1:40 in the ligation buffer to make a ligation solution. Tap off excess wash buffer and add the ligation solution to each well. Incubate for thirty minutes at thirty-seven degrees Celsius.
After ligation incubation, wash twice in Wash Buffer A for two minutes each at room temperature. During washing, make 1x amplification buffer the 5x amplification buffer 1:5 in high purity water.
Dilute the polymerase enzyme 1:80 in the 1x amplification buffer. Tap off excess wash buffer and add the amplification solution to each well. Incubate for one hundred minutes at thirty-seven degrees Celsius. Protect the slides from light from this point forward.
After amplification incubation, wash twice using Wash Buffer B for ten minutes each with gentle orbital shaking at room temperature. Then a final wash in 0.01x concentrated diluted Wash Buffer B for one minute.
To prepare your slide for imaging, mount a coverslip onto your slide using Duolink® PLA mounting media with DAPI. Slowly lower coverslip onto slide to ensure no air bubbles form. Clear nail polish can be used to seal the edges. Wait approximately fifteen minutes before imaging.
After capturing images, analyze results using the Duolink® ImageTool to obtain quantification of PLA signals. Always consider the user guides and our technical support team for additional information on the Duolink® PLA technology
For more information, visit us at sigma-aldrich.com/duolink.