Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Use the antibody diluent to dilute the primary antibodies. Apply 40 microliters of the diluted primary antibody solution per 14 by 14-millimeter space on the slides. Place the slides in a humid chamber, and incubate overnight at 4 degrees Celsius.

In the morning, wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar. Set the Coplin jar on an orbital shaker set to 60 RPM. Place the slides on a paper towel to let wash buffer run off the slide, and gently wipe the edges.

Prepare a 40-microliter solution containing plus and minus probes. Add the solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 1 hour at 37 degrees Celsius. Wash the slides twice for 5 minutes each with 50 milliliters of 1x wash buffer A at room temperature in a Coplin jar.

Set the Coplin jar on an orbital shaker set to 60 RPM. Dilute the ligation buffer 1 to 5 with ultrapure water. Use this buffer to dilute the ligase 1 to 40 to prepare a working stock of ligation solution. Place the slide on a paper towel to let the wash buffer run off the sides, and gently wipe the edges.

Add 40 microliters of the ligation solution to each 14 by 14-millimeter space. Incubate the slides in a humid chamber for 30 minutes at 37 degrees Celsius. After washing and drying the slides as previously described, add 40 microliters of freshly-prepared amplification solution protected from light to each 14 by 14-millimeter space.

Incubate the slides in the humid dark chamber for 1 hour and 40 minutes at 37 degrees Celsius. Wash the slides twice for 10 minutes each with 50 milliliters of 1x wash buffer B, and wash the slides one time for 1 minute with 50 milliliters of 0.01x wash buffer B.

After drawing the epoxy-coated perimeter of the slide, add 10 microliters of mounting medium to each sample, and gently lay a coverslip on top, allowing for the mounting medium to spread out. Paint around the edge of the coverslip with nail polish to seal the coverslip and slide. Avoid moving the coverslip. Let the nail polish harden for at least 20 minutes at room temperature protected from light.